Compositions and methods for the preparation of protease resistant human growth hormone glycosylation mutants

ABSTRACT

The present invention relates to protease resistant mutants of human growth hormone, which contain newly introduced proteolysis resistant mutations and N-linked or O-linked glycosylation site(s), such that these recombinantly produced polypeptides have glycosylation patterns distinctly different from that of the naturally occurring human growth hormone. The polynucleotide coding sequences for the mutants, expression cassettes comprising the coding sequences, cells expressing the mutants, and methods for producing the mutants are also disclosed. Further disclosed are pharmaceutical compositions comprising the mutants and method for using the mutants.

BACKGROUND OF THE INVENTION

Human growth hormone (hGH) and agonist variants thereof are members of afamily of recombinant proteins, described in U.S. Pat. No. 4,658,021 andU.S. Pat. No. 5,633,352. Their recombinant production and methods of useare detailed in U.S. Pat. Nos. 4,342,832, 4,601,980; U.S. Pat. No.4,898,830; U.S. Pat. No. 5,424,199; and U.S. Pat. No. 5,795,745. Humangrowth hormone participates in various aspects of the regulation ofnormal human growth and development. Through interaction with itsreceptors, this 22 kDa pituitary hormone modulates a multitude ofbiological effects, such as linear growth (somatogenesis), lactation,activation of macrophages, and insulin-like and diabetogenic effects.Chawla, Annu. Rev. Med., 34: 519 (1983); Edwards et al., Science, 239:769 (1988); Isaksson et al., Annu. Rev. Physiol., 47: 483 (1985); Thomerand Vance, J. Clin. Invest., 82: 745 (1988); Hughes and Friesen, Annu.Rev. Physiol., 47: 469 (1985).

The administration of glycosylated and non-glycosylated peptides forengendering a particular physiological response is well known in themedicinal arts. Both purified and recombinant hGH have been used fortreating conditions and diseases due to hGH deficiency, e.g., dwarfismin children. A principal factor that has limited the use of therapeuticpeptides is the immunogenic nature of most peptides. In a patient, animmunogenic response to an administered peptide can neutralize thepeptide and/or lead to the development of an allergic response in thepatient. Other deficiencies of therapeutic glycopeptides includesuboptimal potency and rapid clearance rates. The problems inherent inpeptide therapeutics are recognized in the art, and various methods ofeliminating the problems have been investigated. For example, to providesoluble peptide therapeutics, synthetic polymers have been attached tothe peptide backbone.

The attachment of synthetic polymers to the peptide backbone to improvethe pharmacokinetic properties of glycoprotein therapeutics is known inthe art. An exemplary polymer that has been conjugated to peptides ispoly(ethylene glycol) (“PEG”). The use of PEG to derivatize peptidetherapeutics has been demonstrated to reduce the immunogenicity of thepeptides. For example, U.S. Pat. No. 4,179,337 (Davis et al.) disclosesnon-immunogenic polypeptides such as enzymes and peptide hormonescoupled to polyethylene glycol (PEG) or polypropylene glycol. Between 10and 100 moles of polymer are used per mole of polypeptide and at least15% of the physiological activity is maintained. In addition to reducedimmunogenicity, the clearance time in circulation is prolonged due tothe increased size of the PEG-conjugate of the polypeptides in question.

The principal mode of attachment of PEG, and its derivatives, topeptides is a non-specific bonding through a peptide amino acid residue(see e.g., U.S. Pat. No. 4,088,538 U.S. Pat. No. 4,496,689, U.S. Pat.No. 4,414,147, U.S. Pat. No. 4,055,635, and PCT WO 87/00056). Anothermode of PEG-to-peptide attachment is through the non-specific oxidationof glycosyl residues on a glycopeptide (see e.g., WO 94/05332).

In many chemical PEGylation methods, poly(ethyleneglycol) is added in arandom, non-specific manner to reactive residues on a peptide backbone.The random addition of PEG molecules has its inherent disadvantages,including, e.g. the lack of homogeneity in the final product andpotential for reduction in the biological or enzymatic activity of thepeptide. Therefore, a derivitization strategy that results in theformation of a specifically labeled, readily characterizable,essentially homogeneous product is far superior in the context oftherapeutic peptide production. Such methods have been developed.

Specifically labeled, homogeneous peptide therapeutics can be producedin vitro through the action of enzymes. Unlike the typical non-specificmethods for attaching a synthetic polymer or other label to a peptide,enzyme-based syntheses have the advantages of regioselectivity andstereoselectivity. Two principal classes of enzymes that can be employedin the synthesis of labeled peptides are glycosyltransferases (e.g.,sialyltransferases, oligosaccharyltransferases,N-acetylglucosaminyltransferases) and glycosidases. These enzymes can beused for the specific attachment of sugars which can be subsequentlymodified to comprise a therapeutic moiety. Alternatively,glycosyltransferases and modified glycosidases can be used to directlytransfer modified sugars to a peptide backbone (see e.g., U.S. Pat. No.6,399,336, and U.S. Patent Application Publications 20030040037,20040132640, 20040137557, 20040126838, and 20040142856). Methodscombining both chemical and enzymatic synthetic elements are also known(see e.g., Yamamoto et al. Carbohydr. Res. 305: 415-422 (1998) and U.S.Patent Application Publication 20040137557).

In response to the need for improved therapeutic hGH, the presentinvention provides a glycopegylated hGH that is therapeutically activeand which has pharmacokinetic parameters and properties that areimproved relative to an identical, or closely analogous, hGH peptidethat is not glycopegylated. Furthermore, the present invention providescost-effective methods by which improved hGH peptides can be produced onan industrial scale.

Glycosyl residues have also been modified to bear ketone groups. Forexample, Mahal and co-workers (Science 276: 1125 (1997)) have preparedN-levulinoyl mannosamine (“ManLev”), which has a ketone functionality atthe position normally occupied by the acetyl group in the naturalsubstrate. Cells were treated with the ManLev, thereby incorporating aketone group onto the cell surface. See, also Saxon et al., Science 287:2007 (2000); Hang et al., J. Am. Chem. Soc. 123: 1242 (2001); Yarema etal., J. Biol. Chem. 273: 31168 (1998); and Charter et al., Glycobiology10: 1049 (2000).

Carbohydrates are attached to glycopeptides in several ways of whichN-linked to asparagine and mucin-type O-linked to serine and threonineare the most relevant for recombinant glycoprotein therapeutics. Adetermining factor for initiation of glycosylation of a protein is theprimary sequence context, although clearly other factors includingprotein region and conformation have their roles. N-linked glycosylationoccurs at the consensus sequence NXS/T, where X can be any amino acidbut proline.

As previously mentioned, rapid in vivo degradation and clearance rateare other well-known problems that interfere with the optimal desiredphysiological effects of administered polypeptides. Soon afterinjection, polypeptides such as human growth hormone are readilyproteolyzed by numerous proteases in the blood and lymphatic system.These proteases cleave the human growth hormone in both the aminointernal and carboxy terminal regions, thereby fragmenting the proteinand reducing the growth effects of hGH. The proteolysis also facilitatesclearance of the degraded protein, dramatically reducing the residencetime in the body after injection. In light of the above, there is a needfor polypeptides with protease resistance and method of producing suchpolypeptides.

The methods discussed above do not provide access to industriallyrelevant quantities of modified peptides that substantially retain thepharmacological activity of their unmodified analogues and possessprotease resistance.

The present invention answers these needs by providing hGH mutants thatcontain newly introduced O-linked glycosylation sites, providingflexibility in glycosylation and/or glycoconjugation, e.g.,glycoPEGylation of these recombinant hGH mutants. The O-glycosylationmutants optionally further include one or more proteolysis resistantmutation or sites for chemical PEGylation of region(s) most susceptibleto proteases. Moreover, the invention provides an industrially practicalmethod for the modification of N- or O-linked mutant hGH peptides withmodifying groups such as water-soluble polymers, therapeutic moieties,biomolecules, and the like. Of particular interest are methods in whichthe modified mutant hGH has improved properties, which enhance its useas a therapeutic or diagnostic agent.

SUMMARY OF THE INVENTION

The present invention provides for hGH mutants with N-linked or O-linkedglycosylation sites not found in wild-type hGH. Exemplary embodiments ofthe invention include N- or O-linked glycosylated hGH mutants having oneor more characteristics selected from the following: glycoPEGylation,protease resistance, and chemically PEGylation. Through the controlledmodification of hGH, the present invention yields novel hGH derivativeswith pharmacokinetic properties that are improved relative to thecorresponding native hGH.

In a first aspect, the present invention provides an isolated nucleicacid comprising a polynucleotide sequence encoding a mutant human growthhormone. The mutant human growth hormone comprises an N-linked orO-linked glycosylation site and/or proteolysis resistant mutation(s)that are not present in wild-type human growth hormone. In exemplaryembodiments, the wild-type human growth hormones have the amino acidsequence of pituitary-derived GH-N (SEQ ID NO:1) or placenta-derivedGH-V (SEQ ID NO:2). In some preferred embodiments, the mutant humangrowth hormone includes the amino acid sequence of SEQ ID NO:3, 4, 5, 6,7, 8, 9, 80, 81, 82, 83, 84, 85, 86, 87, and 88.

In another aspect, the present invention provides an expression cassetteor a cell that comprises a nucleic acid, e.g., an isolated nucleic acid,including a polynucleotide sequence encoding a mutant human growthhormone. The mutant human growth hormone includes an N-linked orO-linked glycosylation site and/or proteolysis inhibiting mutation(s)that are not present in the wild-type human growth hormone.

In still another aspect, the present invention provides a mutantglycoPEGylated human growth hormone that includes one or more N-linkedor O-linked glycosylation site not present in the wild-type human growthhormone. In some embodiments, the wild-type human growth hormone has theamino acid sequence of SEQ ID NO:1 or SEQ ID NO:2. In some preferredembodiments, the mutant human growth hormone comprises the amino acidsequence of SEQ ID NO:3, 4, 5, 6, 7, 8, 9, 80, 81, 82, 83, 84, 85, 86,87, and 88.

In still another aspect, the present invention provides a mutant humangrowth hormone that includes one or more N-linked or O-linkedglycosylation site not present in the wild-type human growth hormone andone or more proteolysis resistant mutations(s) not present in thewild-type human growth hormone. In some embodiments, the wild-type humangrowth hormone has the amino acid sequence of SEQ ID NO:1 or SEQ IDNO:2. In some preferred embodiments, the mutant human growth hormonecomprises the amino acid sequence of SEQ ID NO:3, 4, 5, 6, 7, 8, 9, 80,81, 82, 83, 84, 85, 86, 87, and 88.

In still another aspect, the present invention provides a chemicallyPEGylated mutant human growth hormone that includes one or more N-linkedor O-linked glycosylation site not present in the wild-type human growthhormone. In some embodiments, the wild-type human growth hormone has theamino acid sequence of SEQ ID NO:1 or SEQ ID NO:2. In some preferredembodiments, the mutant human growth hormone comprises the amino acidsequence of SEQ ID NO:3, 4, 5, 6, 7, 8, 9, 80, 81, 82, 83, 84, 85, 86,87, and 88.

In another aspect, the present invention provides a glycoPEGylatedmutant human growth hormone that includes one or more N-linked orO-linked glycosylation site not present in the wild-type human growthhormone and one or more proteolysis resistant mutations(s) not presentin the wild-type human growth hormone. In some embodiments, thewild-type human growth hormone has the amino acid sequence of SEQ IDNO:1 or SEQ ID NO:2. In some preferred embodiments, the mutant humangrowth hormone comprises the amino acid sequence of SEQ ID NO:3, 4, 5,6, 7, 8, 9, 80, 81, 82, 83, 84, 85, 86, 87, and 88.

In another aspect, the present invention provides a chemically PEGylatedmutant human growth hormone that includes one or more N-linked orO-linked glycosylation site not present in the wild-type human growthhormone and one or more proteolysis resistant mutations(s) not presentin the wild-type human growth hormone. In some embodiments, thewild-type human growth hormone has the amino acid sequence of SEQ IDNO:1 or SEQ ID NO:2. In some preferred embodiments, the mutant humangrowth hormone comprises the amino acid sequence of SEQ ID NO:3, 4, 5,6, 7, 8, 9, 80, 81, 82, 83, 84, 85, 86, 87, and 88.

In another aspect, the present invention provides a glycoPEGylated andchemically PEGylated mutant human growth hormone that includes one ormore N-linked or O-linked glycosylation site not present in thewild-type human growth hormone. In some embodiments, the wild-type humangrowth hormone has the amino acid sequence of SEQ ID NO:1 or SEQ IDNO:2. In some preferred embodiments, the mutant human growth hormonecomprises the amino acid sequence of SEQ ID NO:3, 4, 5, 6, 7, 8, 9, 80,81, 82, 83, 84, 85, 86, 87, and 88.

In another aspect, the present invention provides a glycoPEGylated andchemically PEGylated mutant human growth hormone that includes one ormore N-linked or O-linked glycosylation site not present in thewild-type human growth hormone and one or more proteolysis resistantmutations(s) not present in the wild-type human growth hormone. In someembodiments, the wild-type human growth hormone has the amino acidsequence of SEQ ID NO:1 or SEQ ID NO:2. In some preferred embodiments,the mutant human growth hormone comprises the amino acid sequence of SEQID NO:3, 4, 5, 6, 7, 8, 9, 80, 81, 82, 83, 84, 85, 86, 87, and 88.

In another aspect, the present invention provides a method for making amutant human growth hormone that includes an N-linked or O-linkedglycosylation site and/or proteolysis inhibiting mutation(s) that arenot present in the wild-type human growth hormone. This method includesthe steps of recombinantly producing the mutant human growth hormone,and glycosylating the mutant human growth hormone at the newglycosylation site. In some embodiments, the wild-type human growthhormone has the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2. Insome preferred embodiments, the mutant human growth hormone comprisesthe amino acid sequence of SEQ ID NO:3, 4, 5, 6, 7, 8, 9, 80, 81, 82,83, 84, 85, 86, 87, and 88.

In still a further aspect, the present invention provides apharmaceutical composition having a therapeutically effective amount ofa mutant human growth hormone that includes an N-linked or O-linkedglycosylation site and/or proteolysis inhibiting mutation(s) not presentin the wild-type human growth hormone. In some embodiments, thewild-type human growth hormone has the amino acid sequence of SEQ IDNO:1 or SEQ ID NO:2. In some preferred embodiments, the mutant humangrowth hormone comprises the amino acid sequence of SEQ ID NO:3, 4, 5,6, 7, 8, 9, 80, 81, 82, 83, 84, 85, 86, 87, and 88.

In another aspect, the present invention provides a method for treatinghuman growth hormone deficiency in a subject. The method includesadministering to the subject an amount of a mutant human growth hormoneeffective to treat or ameliorate the growth hormone deficiency. Themutant human growth hormone used in this method comprises an N-linked orO-linked glycosylation site and/or proteolysis inhibiting mutation(s)that do not exist in the corresponding wild-type human growth hormone.In some embodiments, the corresponding wild-type human growth hormonehas the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2. In somepreferred embodiments, the mutant human growth hormone comprises theamino acid sequence of SEQ ID NO:3, 4, 5, 6, 7, 8, 9, 80, 81, 82, 83,84, 85, 86, 87, and 88.

In each of the aspects described above, the mutant human growth hormoneis optionally conjugated to one or more modifying groups, preferably viaglycoconjugation, giving rise to a glycosyl linking group between theglycosylation site and the modifying group. An exemplary modifying groupis poly(ethylene glycol), otherwise known as PEG.

In exemplary embodiments of the present invention, “glycopegylated” hGHmolecules are produced by the enzyme-mediated formation of a conjugatebetween a glycosylated or non-glycosylated hGH peptide and anenzymatically transferable saccharyl moiety that includes one or morepoly(ethylene glycol) moieties within its structure The PEG moiety isattached to the saccharyl moiety directly (i.e., through a single groupformed by the reaction of two reactive groups) or through a linkermoiety, e.g., substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, etc.

Other objects, aspects, and advantages of the present invention will beapparent from the detailed description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 are glycoPEGylation schemes for insect cell and mammalian cellproduced hGH N-linked glycan mutants.

FIG. 2 shows a pituitary derived hGH (GH-N) sequence into which six (6)different exemplary O-linked glycosylation sites have been introduced(SEQ ID NO:90). The wild-type amino acid sequence for GH-N (SEQ ID NO:1)is also shown for comparison. The arrows indicate the threonine residueof the GH-N glycan mutant on which O-linked glycosylation will occur.

FIG. 3 includes the alignment of amino acid sequences for hGH O-linkedGH-N mutant 134(rtg)→ttt (SEQ ID NO:91) and hGH O-linked 5′ GH-N mutant(SEQ ID NO:92) in which amino acids −3 to −1 (ptt) are inserted at theamino terminus, resulting in a 194 amino acid hGH polypeptide, with thenative amino acid sequence of mature hGH-N1 (SEQ ID NO: 1).

FIG. 4 includes the amino acid sequences of hGH O-linked GH-N mutant134(rtg)→ttg (SEQ ID NO:93) and hGH O-linked 5′ GH-N mutant (SEQ IDNO:94) in which amino acids −3 to −1 (mvt) are inserted at the aminoterminus, resulting in a 194 amino acid hGH polypeptide, with the nativeamino acid sequence of mature hGH-N1 (SEQ ID NO:1).

FIG. 5A depicts the amino acid sequence of mature pituitary-derivedhuman growth hormone (GH-N) (SEQ ID NO:1). FIG. 5B depicts the aminoacid sequence of mature placenta-derived human growth hormone (GH-V)(SEQ ID NO:2). FIG. 5C depicts the amino acid sequence of human growthhormone mutant 1 (SEQ ID NO:3). FIG. 5D depicts the amino acid sequenceof human growth hormone mutant 2 (SEQ ID NO:4). FIG. 5E depicts theamino acid sequence of human growth hormone mutant 3 (SEQ ID NO:5). FIG.5F depicts the amino acid sequence of human growth hormone mutant 4 (SEQID NO:6). FIG. 5G depicts the amino acid sequence of human growthhormone mutant 5 (SEQ ID NO:7). FIG. 5H depicts the amino acid sequenceof human growth hormone mutant 6 (SEQ ID NO:8). FIG. 5I depicts theamino acid sequence of human growth hormone mutant 7 (SEQ ID NO:9).

FIG. 6A depicts the amino acid sequence of human growth hormone mutant 8(SEQ ID NO:80). FIG. 6B depicts the amino acid sequence of human growthhormone mutant 9 (SEQ ID NO:81). FIG. 6C depicts the amino acid sequenceof human growth hormone mutant 10 (SEQ ID NO:82). FIG. 6D depicts theamino acid sequence of human growth hormone mutant 11 (SEQ ID NO:83).FIG. 6E depicts the amino acid sequence of human growth hormone mutant12 (SEQ ID NO:84). FIG. 6F depicts the amino acid sequence of humangrowth hormone mutant 13 (SEQ ID NO:85). FIG. 6G depicts the amino acidsequence of human growth hormone mutant 14 (SEQ ID NO:86). FIG. 6Hdepicts the amino acid sequence of human growth hormone mutant 15 (SEQID NO:87). FIG. 6I depicts the amino acid sequence of human growthhormone mutant 16 (SEQ ID NO:88). FIG. 6J depicts the amino acidsequence of human growth hormone mutant 17 (SEQ ID NO:83). FIG. 6Kdepicts the amino acid sequence of mature human growth hormone (GH-N)with the various proteolysis inhibiting mutation sites in bold (SEQ IDNO:95).

FIG. 7 shows data for various hGH mutants with proteolysis inhibitingmutations from an Nb2-11 cell proliferation assay.

FIG. 8 depicts exemplary reactions through which glycoPEGylation ofmutant hGH can be achieved.

FIG. 9 shows the gel runs for the starting material and product in theglycoPEGylation of hGH mutants ID# AI and AG (P¹³⁴TQGAM and P¹⁴⁰TQA).

FIG. 10 shows the gel runs for the starting material and product in theglycoPEGylation of hGH mutant ID# AO (P¹³⁴TINTIAN).

FIG. 11 shows the results of an SDS PAGE silver stain to demonstrate thepurity of four exemplary hGH-PEG conjugates.

FIG. 12 shows the results of an Nb2-11 cell proliferation assay forseven hGH-PEG mutants relative to wild-type hGH.

FIG. 13 shows the respective pharmacokinetic data in rats givensubcutaneous administration of five hGH-PEG mutants (P¹³⁴TINT).

FIG. 14 shows the respective pharmacokinetic data in rats givensubcutaneous administration of three hGH-PEG mutants (P¹⁴⁰TQA).

FIG. 15 shows the respective amount of weight gained byhypophysectomized rats given subcutaneous administration of five hGH-PEGmutants (P¹³⁴TINT).

FIG. 16 shows the respective amount of weight gained byhypophysectomized rats given subcutaneous administration of threehGH-PEG mutants with the P¹⁴⁰TQA mutation motif and 1 hGH-PEG mutantwith a P³TEIP mutation motif.

FIG. 17 shows the respective serum IGF-1 levels in hypophysectomizedrats given subcutaneous administration of two hGH-PEG mutants(P¹³⁴TINT).

FIG. 18 shows the respective serum IGF-1 levels in hypophysectomizedrats given subcutaneous administration of three hGH-PEG mutants(P¹³⁴TINT).

FIG. 19 shows the respective serum IGF-1 levels in hypophysectomizedrats given subcutaneous administration of three hGH-PEG mutants with theP¹⁴⁰TQA mutation motif and 1 hGH-PEG mutant with a P³TEIP mutationmotif.

FIG. 20 shows the respective weight gain and respective serum IGF-1levels over time in hypophysectomized rats that were administered arecombinant wild-type hGH and an exemplary hGH mutant at varyingdosages.

FIG. 21 shows the respective weight gain and respective serum IGF-1levels over time in hypophysectomized rats that were administered arecombinant wild-type hGH and various exemplary hGH mutants.

FIG. 22 shows the respective mean hGH concentration levels over time inrats that were administered single subcutaneous injections of arecombinant wild-type hGH and various exemplary hGH mutants.

FIG. 23 shows an exemplary hGH mutant construct with proteaserecognition sites indicated in block shading and exemplary mutationsindicated in bold lettering in the upper panel. In the lower panel,mutations in an exemplary hGH mutant construct are indicated in boldunderlined lettering and exemplary sites for chemical PEGylation areindicated in bold, encircled lettering.

FIG. 24 are SDS-PAGE analysis results for two exemplary glycoPEGylatedhGH, illustrating the extent to which two exemplary glycoPEGylated hGHmutants are chemically PEGylated over 18 hours.

FIG. 25 shows the results of an Nb2-11 cell proliferation assay forseven hGH-PEG mutants relative to wild-type hGH.

DETAILED DESCRIPTION OF THE INVENTION

Abbreviations

PEG, poly(ethylene glycol), e.g. monomethoxy poly(ethyleneglycol)(m-PEG); PPG, poly(propyleneglycol); Ara, arabinosyl; Fru, fiuctosyl;Fuc, fucosyl; Gal, galactosyl; GalNAc, N-acetylgalactosaminyl; Glc,glucosyl; GlcNAc, N-acetylglucosaminyl; Man, mannosyl; ManAc,N-acetylmannosaminyl; Xyl, xylosyl; NeuAc, sialyl or N-acetylneuraminyl;Sia, sialyl or N-acetylneuraminyl, and derivatives and analoguesthereof.

Definitions

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Generally,the nomenclature used herein and the laboratory procedures in cellculture, molecular genetics, organic chemistry and nucleic acidchemistry and hybridization are those well known and commonly employedin the art. Standard techniques are used for nucleic acid and peptidesynthesis. The techniques and procedures are generally performedaccording to conventional methods in the art and various generalreferences (see generally, Sambrook et al. MOLECULAR CLONING: ALABORATORY MANUAL, 2d ed. (1989) Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., which is incorporated herein by reference),which are provided throughout this document. The nomenclature usedherein and the laboratory procedures in analytical chemistry, andorganic synthetic described below are those well known and commonlyemployed in the art. Standard techniques, or modifications thereof, areused for chemical syntheses and chemical analyses.

The term “nucleic acid” or “polynucleotide” refers to deoxyribonucleicacids (DNA) or ribonucleic acids (RNA) and polymers thereof in eithersingle- or double-stranded form. Unless specifically limited, the termencompasses nucleic acids containing known analogues of naturalnucleotides that have similar binding properties as the referencenucleic acid and are metabolized in a manner similar to naturallyoccurring nucleotides. Unless otherwise indicated, a particular nucleicacid sequence also implicitly encompasses conservatively modifiedvariants thereof (e.g., degenerate codon substitutions), alleles,orthologs, SNPs, and complementary sequences as well as the sequenceexplicitly indicated. Specifically, degenerate codon substitutions maybe achieved by generating sequences in which the third position of oneor more selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991);Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini etal., Mol. Cell. Probes 8:91-98 (1994)). The term nucleic acid is usedinterchangeably with gene, cDNA, and mRNA encoded by a gene.

The term “gene” means the segment of DNA involved in producing apolypeptide chain. It may include regions preceding and following thecoding region (leader and trailer) as well as intervening sequences(introns) between individual coding segments (exons).

The term “isolated,” when applied to a nucleic acid or protein, denotesthat the nucleic acid or protein is essentially free of other cellularcomponents with which it is associated in the natural state. It ispreferably in a homogeneous state although it can be in either a dry oraqueous solution. Purity and homogeneity are typically determined usinganalytical chemistry techniques such as polyacrylamide gelelectrophoresis or high performance liquid chromatography. A proteinthat is the predominant species present in a preparation issubstantially purified. In particular, an isolated gene is separatedfrom open reading frames that flank the gene and encode a protein otherthan the gene of interest. The term “purified” denotes that a nucleicacid or protein gives rise to essentially one band in an electrophoreticgel. Particularly, it means that the nucleic acid or protein is at least85% pure, more preferably at least 95% pure, and most preferably atleast 99% pure.

The term “proximate,” when not used in reference to a proline residue,describes an amino acid having five or few amino acids removed from theC terminal end or N terminal end of the protease recognition site.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an α carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds having a structure that is different from the generalchemical structure of an amino acid, but that functions in a mannersimilar to a naturally occurring amino acid. As used herein, “aminoacid,” whether it is in a linker or a component of a peptide sequencerefers to both the D- and L-isomer of the amino acid as well as mixturesof these two isomers.

There are various known methods in the art that permit the incorporationof an unnatural amino acid derivative or analog into a polypeptide chainin a site-specific manner, see, e.g., WO 02/086075.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, “conservatively modified variants” refers to those nucleicacids that encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein that encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidthat encodes a polypeptide is implicit in each described sequence.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The following eight groups each contain amino acids that areconservative substitutions for one another:

Alanine (A), Glycine (G);

Aspartic acid (D), Glutamic acid (E);

Asparagine (N), Glutamine (Q);

Arginine (R), Lysine (K);

Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

Serine (S), Threonine (T); and

Cysteine (C), Methionine (M)

(see, e.g., Creighton, Proteins (1984)).

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

In the present application, amino acid residues are numbered accordingto their relative positions from the left most residue, which isnumbered 1, in an unmodified wild-type polypeptide sequence.

“Proximate a proline residue,” as used herein refers to an amino acidthat is less than about 10 amino acids removed from a proline residue,preferably, less than about 9, 8, 7, 6 or 5 amino acids removed from aproline residue, more preferably, less than about 4, 3, 2 or 1 residuesremoved from a proline residue. The amino acid “proximate a prolineresidue” may be on the C- or N-terminal side of the proline residue.

The term “sialic acid” refers to any member of a family of nine-carboncarboxylated sugars. The most common member of the sialic acid family isN-acetyl-neuraminic acid(2-keto-5-acetamido-3,5-dideoxy-D-glycero-D-galactononulopyranos-1-onicacid (often abbreviated as Neu5Ac, NeuAc, or NANA). A second member ofthe family is N-glycolyl-neuraminic acid (Neu5Gc or NeuGc), in which theN-acetyl group of NeuAc is hydroxylated. A third sialic acid familymember is 2-keto-3-deoxy-nonulosonic acid (KDN) (Nadano et al. (1986) J.Biol. Chem. 261: 11550-11557; Kanamori et al., J. Biol. Chem. 265:21811-21819 (1990)). Also included are 9-substituted sialic acids suchas a 9-O—C₁-C₆ acyl-Neu5Ac like 9-O-lactyl-Neu5Ac or 9-O-acetyl-Neu5Ac,9-deoxy-9-fluoro-Neu5Ac and 9-azido-9-deoxy-Neu5Ac. For review of thesialic acid family, see, e.g., Varki, Glycobiology 2: 25-40 (1992);Sialic Acids: Chemistry, Metabolism and Function, R. Schauer, Ed.(Springer-Verlag, New York (1992)). The synthesis and use of sialic acidcompounds in a sialylation procedure is disclosed in internationalapplication WO 92/16640, published Oct. 1, 1992.

“Peptide” refers to a polymer in which the monomers are amino acids andare joined together through amide bonds, alternatively referred to as apolypeptide or a protein. Additionally, unnatural amino acids, forexample, β-alanine, phenylglycine and homoarginine are also included.Amino acids that are not gene-encoded may also be used in the presentinvention. Furthermore, amino acids that have been modified to includereactive groups, glycosylation sites, polymers, therapeutic moieties,biomolecules and the like may also be used in the invention. All of theamino acids used in the present invention may be either the d- or1-isomer. The 1-isomer is generally preferred. In addition, otherpeptidomimetics are also useful in the present invention. As usedherein, “peptide” refers to both glycosylated and unglycosylatedpeptides. Also included are peptides that are incompletely glycosylatedby a system that expresses the peptide. For a general review, see,Spatola, A. F., in Chemistry and Biochemistry of Amino Acids, Peptidesand Proteins, B. Weinstein, eds., Marcel Dekker, New York, p. 267(1983). The numbering of the amino acid residues in the peptides of theinvention is based on the initial unmodified sequence in which the leftmost residue, methionine, is numbered as position 1.

The term “peptide conjugate,” refers to species of the invention inwhich a peptide is conjugated with a modified sugar as set forth herein.

hGH peptides of the invention comprise sequences with additional aminoacids. These amino acids can be inserted into the middle of thesequence, as shown in “O-linked Glycosylation of Peptides”, filed Jan.10, 2005, Atty. Docket No. 040853-01-5138-US. Additional amino acids,both natural and unnatural, can also be attached at the beginning or endof the amino acid sequence.

As used herein, the term “proteolysis resistant mutant” or “proteaseresistant mutant” refers to a hGH mutant that is proteolyzed more slowlythan the corresponding wild-type hGH. In preferred embodiments, theprotease resistant mutant of the invention is proteolyzed at a rate of90% or less relative to the corresponding wild-type hGH. Morepreferably, the protease resistant mutant of the invention isproteolyzed at a rate of 70% or less relative to the correspondingwild-type hGH. Even more preferably, the protease resistant mutant ofthe invention is proteolyzed at a rate of 50% or less relative to thecorresponding wild-type hGH.

The term “mutating” or “mutation,” as used in the context of introducingadditional N- or O-linked glycosylation site(s) into a wild-type humangrowth hormone, refers to the deletion, insertion, or substitution ofany nucleotide or amino acid residue, by chemical, enzymatic, or anyother means, in a polynucleotide sequence encoding a wild-type humangrowth hormone or the amino acid sequence of a wild-type human growthhormone, respectively, such that the amino acid sequence of theresulting human growth hormone comprises at least one N- or O-linkedglycosylation site that does not exist in the corresponding wild-typehuman growth hormone. In the case of amino acid substitution, bothconservative and non-conservative substitutions may be used to create ahGH mutant that contains a new N- or O-linked glycosylation site.

The site for a mutation introducing a new N- or O-linked glycosylationsite may be located anywhere in the polypeptide. Exemplary amino acidsequences for human growth hormone mutants are depicted in SEQ IDNOs:3-9, and 80-89. A “mutant human growth hormone” of this inventionthus comprises at least one mutated amino acid residue. On the otherhand, the wild-type human growth hormone whose coding sequence ismodified to generate a mutant human growth hormone is referred to inthis application as “the corresponding wild-type human growth hormone.”For example, SEQ ID NO:1 is the amino acid sequence of the correspondingwild-type human growth hormone for mutant human growth hormones havingthe amino acid sequences of SEQ ID NOs:3-9, and 80-89.

As used herein, the term “modified sugar,” refers to a naturally- ornon-naturally-occurring carbohydrate that is enzymatically added onto anamino acid or a glycosyl residue of a peptide in a process of theinvention. The modified sugar is selected from a number of enzymesubstrates including, but not limited to sugar nucleotides (mono-, di-,and tri-phosphates), activated sugars (e.g., glycosyl halides, glycosylmesylates) and sugars that are neither activated nor nucleotides. Insome embodiments, the “modified sugar” can be covalently functionalizedwith a “modifying group.”

As used herein, the term “modifying group” refers to a component of thehGH conjugate that is covalently attached to a glycosyl linking group. Amodifying group can be a component of the modified sugar that issubsequently attached to the hGH peptide. A modifying group can also beattached directly to a sugar moiety that is already attached to the hGHpeptide. Useful modifying groups include, but are not limited to,water-soluble polymer moieties such as PEG, water-insoluble polymermoieties, therapeutic moieties, diagnostic moieties, biomolecules, andthe like. The modifying group also includes reactive functional groups,such as levulinic acid. These reactive functional groups can serve asthe locus of attachment for water-soluble polymers such as PEG moieties,therapeutic moieties, diagnostic moieties, biomolecules, and the like.These reactive functional groups can also comprise protecting groupswhich can be removed at appropriate times to facilitate properfunctionalization. Reactive functional groups with protecting groups arealternatively known as masked reactive functional groups. The modifyinggroup is preferably not a naturally occurring, or an unmodifiedcarbohydrate. The locus of functionalization with the modifying group isselected such that it does not prevent the “modified sugar” from beingadded enzymatically to a peptide.

The term “water-soluble” refers to moieties that have some detectabledegree of solubility in water. Methods to detect and/or quantify watersolubility are well known in the art. Exemplary water-soluble polymersinclude peptides, saccharides, poly(ethers), poly(amines),poly(carboxylic acids) and the like. Peptides can have mixed sequencesor be composed of a single amino acid, e.g., poly(lysine). An exemplarypolysaccharide is poly(sialic acid). An exemplary poly(ether) ispoly(ethylene glycol). Poly(ethylene imine) is an exemplary polyamine,and poly(acrylic) acid is a representative poly(carboxylic acid).

The polymer backbone of the water-soluble polymer can be poly(ethyleneglycol) (i.e. PEG). However, it should be understood that other relatedpolymers are also suitable for use in the practice of this invention andthat the use of the term PEG or poly(ethylene glycol) is intended to beinclusive and not exclusive in this respect. The term PEG includespoly(ethylene glycol) in any of its forms, including alkoxy PEG,difunctional PEG, multiarmed PEG, forked PEG, branched PEG, pendent PEG(i.e. PEG or related polymers having one or more functional groupspendent to the polymer backbone), or PEG with degradable linkagestherein.

The polymer backbone can be linear or branched. Branched polymerbackbones are generally known in the art. Typically, a branched polymerhas a central branch core moiety and a plurality of linear polymerchains linked to the central branch core. PEG is commonly used inbranched forms that can be prepared by addition of ethylene oxide tovarious polyols, such as glycerol, pentaerythritol and sorbitol. Thecentral branch moiety can also be derived from several amino acids, suchas lysine. The branched poly(ethylene glycol) can be represented ingeneral form as R(-PEG-OH)_(m) in which R represents the core moiety,such as glycerol or pentaerythritol, and m represents the number ofarms. Multi-armed PEG molecules, such as those described in U.S. Pat.No. 5,932,462, which is incorporated by reference herein in itsentirety, can also be used as the polymer backbone.

Many other polymers are also suitable for the invention. Polymerbackbones that are non-peptidic and water-soluble, with from 2 to about300 termini, are particularly useful in the invention. Examples ofsuitable polymers include, but are not limited to, other poly(alkyleneglycols), such as poly(propylene glycol) (“PPG”), copolymers of ethyleneglycol and propylene glycol and the like, poly(oxyethylated polyol),poly(olefinic alcohol), poly(vinylpyrrolidone),poly(hydroxypropylmethacrylamide), poly(α-hydroxy acid), poly(vinylalcohol), polyphosphazene, polyoxazoline, poly(N-acryloylmorpholine),such as described in U.S. Pat. No. 5,629,384, which is incorporated byreference herein in its entirety, and copolymers, terpolymers, andmixtures thereof. Although the molecular weight of each chain of thepolymer backbone can vary, it is typically in the range of from about100 Da to about 100,000 Da, often from about 6,000 Da to about 80,000Da.

The “area under the curve” or “AUC”, as used herein in the context ofadministering a peptide drug to a patient, is defined as total areaunder the curve that describes the concentration of drug in systemiccirculation in the patient as a function of time from zero to infinity.

The term “half-life” or “t½”, as used herein in the context ofadministering a peptide drug to a patient, is defined as the timerequired for plasma concentration of a drug in a patient to be reducedby one half. There may be more than one half-life associated with thepeptide drug depending on multiple clearance mechanisms, redistribution,and other mechanisms well known in the art. Usually, alpha and betahalf-lives are defined such that the alpha phase is associated withredistribution, and the beta phase is associated with clearance.However, with protein drugs that are, for the most part, confined to thebloodstream, there can be at least two clearance half-lives. For someglycosylated peptides, rapid beta phase clearance may be mediated viareceptors on macrophages, or endothelial cells that recognize terminalgalactose, N-acetylgalactosamine, N-acetylglucosamine, mannose, orfucose. Slower beta phase clearance may occur via renal glomerularfiltration for molecules with an effective radius<2 nm (approximately 68kD) and/or specific or non-specific uptake and metabolism in tissues.GlycoPEGylation may cap terminal sugars (e.g., galactose orN-acetylgalactosamine) and thereby block rapid alpha phase clearance viareceptors that recognize these sugars. It may also confer a largereffective radius and thereby decrease the volume of distribution andtissue uptake, thereby prolonging the late beta phase. Thus, the preciseimpact of glycoPEGylation on alpha phase and beta phase half-lives willvary depending upon the size, state of glycosylation, and otherparameters, as is well known in the art. Further explanation of“half-life” is found in Pharmaceutical Biotechnology (1997, DFACrommelin and RD Sindelar, eds., Harwood Publishers, Amsterdam, pp101-120).

The term “glycoconjugation,” as used herein, refers to the enzymaticallymediated conjugation of a modified sugar species to an amino acid orglycosyl residue of a polypeptide, e.g., a mutant human growth hormoneof the present invention. A subgenus of “glycoconjugation” is“glycoPEGylation,” in which the modifying group of the modified sugar ispoly(ethylene glycol), and alkyl derivative (e.g., m-PEG) or reactivederivative (e.g., H2N-PEG, HOOC-PEG) thereof. Exemplary methods ofglycoconjugation are described in PCT/JUS02/32263, U.S. application Ser.No. 10/411,012.

The terms “large-scale” and “industrial-scale” are used interchangeablyand refer to a reaction cycle that produces at least about 250 mg,preferably at least about 500 mg, and more preferably at least about 1gram of glycoconjugate at the completion of a single reaction cycle.

The term, “glycosyl linking group,” as used herein refers to a glycosylresidue to which a modifying group (e.g., PEG moiety, therapeuticmoiety, biomolecule) is covalently attached; the glycosyl linking groupjoins the modifying group to the remainder of the conjugate. In themethods of the invention, the “glycosyl linking group” becomescovalently attached to a glycosylated or unglycosylated peptide, therebylinking the agent to an amino acid and/or glycosyl residue on thepeptide. A “glycosyl linking group” is generally derived from a“modified sugar” by the enzymatic attachment of the “modified sugar” toan amino acid and/or glycosyl residue of the peptide. The glycosyllinking group can be a saccharide-derived structure that is degradedduring formation of modifying group-modified sugar cassette (e.g.,oxidation-Schiff base formation-reduction), or the glycosyl linkinggroup may be intact. An “intact glycosyl linking group” refers to alinking group that is derived from a glycosyl moiety in which thesaccharide monomer that links the modifying group to the remainder ofthe conjugate is not degraded, e.g., oxidized by sodium metaperiodate.“Intact glycosyl linking groups” of the invention may be derived from anaturally occurring oligosaccharide by addition of glycosyl unit(s) orremoval of one or more glycosyl unit from a parent saccharide structure.

The term “targeting moiety,” as used herein, refers to species that willselectively localize in a particular tissue or region of the body. Thelocalization is mediated by specific recognition of moleculardeterminants, molecular size of the targeting agent or conjugate, ionicinteractions, hydrophobic interactions and the like. Other mechanisms oftargeting an agent to a particular tissue or region are known to thoseof skill in the art. Exemplary targeting moieties include antibodies,antibody fragments, transferrin, HS-glycoprotein, coagulation factors,serum proteins, β-glycoprotein, hGH, GM-CSF, M-CSF, EPO and the like.

As used herein, “therapeutic moiety” means any agent useful for therapyincluding, but not limited to, antibiotics, anti-inflammatory agents,anti-tumor drugs, cytotoxins, and radioactive agents. “Therapeuticmoiety” includes prodrugs of bioactive agents, constructs in which morethan one therapeutic moiety is bound to a carrier, e.g., multivalentagents. Therapeutic moiety also includes proteins and constructs thatinclude proteins. Exemplary proteins include, but are not limited to,Erythropoietin (EPO), Granulocyte Colony Stimulating Factor (G-CSF),Granulocyte Macrophage Colony Stimulating Factor (GMCSF), Interferon(e.g., Interferon-α, -β, -γ), Interleukin (e.g., Interleukin II), serumproteins (e.g., Factors VII, VIIa, VIII, IX, and X), Human ChorionicGonadotropin (HCG), Follicle Stimulating Hormone (FSH) and LutenizingHormone (LH) and antibody fusion proteins (e.g. Tumor Necrosis FactorReceptor ((TNFR)/Fc domain fusion protein)).

As used herein, “a radioactive agent” includes any radioisotope that iseffective in diagnosing or destroying a tumor. Examples include, but arenot limited to, indium-111, cobalt-60. Additionally, naturally occurringradioactive elements such as uranium, radium, and thorium, whichtypically represent mixtures of radioisotopes, are suitable examples ofa radioactive agent. The metal ions are typically chelated with anorganic chelating moiety.

Many useful chelating groups, crown ethers, cryptands and the like areknown in the art and can be incorporated into the compounds of theinvention (e.g., EDTA, DTPA, DOTA, NTA, HDTA, etc. and their phosphonateanalogs such as DTPP, EDTP, HDTP, NTP, etc). See, for example, Pitt etal., “The Design of Chelating Agents for the Treatment of IronOverload,” In, INORGANIC CHEMISTRY IN BIOLOGY AND MEDICINE; Martell,Ed.; American Chemical Society, Washington, D.C., 1980, pp. 279-312;Lindoy, THE CHEMISTRY OF MACROCYCLIC LIGAND COMPLEXES; CambridgeUniversity Press, Cambridge, 1989; Dugas, BIOORGANIC CHEMISTRY;Springer-Verlag, New York, 1989, and references contained therein.

Additionally, a manifold of routes allowing the attachment of chelatingagents, crown ethers and cyclodextrins to other molecules is availableto those of skill in the art. See, for example, Meares et al.,“Properties of In vivo Chelate-Tagged Proteins and Polypeptides.” In,MODIFICATION OF PROTEINS: FOOD, NUTRITIONAL, AND PHARMACOLOGICALASPECTS;” Feeney, et al., Eds., American Chemical Society, Washington,D.C., 1982, pp. 370-387; Kasina et al., Bioconjugate Chem., 9: 108-117(1998); Song et al., Bioconjugate Chem., 8: 249-255 (1997).

As used herein, “pharmaceutically acceptable carrier” includes anymaterial, which when combined with the conjugate retains the conjugate'sactivity and is non-reactive with the subject's immune systems. Examplesinclude, but are not limited to, any of the standard pharmaceuticalcarriers such as a phosphate buffered saline solution, water, emulsionssuch as oil/water emulsion, and various types of wetting agents. Othercarriers may also include sterile solutions, tablets including coatedtablets and capsules. Typically such carriers contain excipients such asstarch, milk, sugar, certain types of clay, gelatin, stearic acid orsalts thereof, magnesium or calcium stearate, talc, vegetable fats oroils, gums, glycols, or other known excipients. Such carriers may alsoinclude flavor and color additives or other ingredients. Compositionscomprising such carriers are formulated by well known conventionalmethods.

As used herein, “administering,” means oral administration, inhalation,administration as a suppository, topical contact, intravenous,intraperitoneal, intramuscular, intralesional, intranasal orsubcutaneous administration, or the implantation of a slow-releasedevice e.g., a mini-osmotic pump, to the subject. Administration is byany route including parenteral, and transmucosal (e.g., oral, nasal,vaginal, rectal, or transdermal). Parenteral administration includes,e.g., intravenous, intramuscular, intra-arteriole, intradermal,subcutaneous, intraperitoneal, intraventricular, and intracranial. Othermodes of delivery include, but are not limited to, the use of liposomalformulations, intravenous infusion, transdermal patches, etc.

The term “ameliorating” or “ameliorate” refers to any indicia of successin the treatment of a pathology or condition, including any objective orsubjective parameter such as abatement, remission or diminishing ofsymptoms or an improvement in a patient's physical or mental well-being.Amelioration of symptoms can be based on objective or subjectiveparameters, including the results of a physical examination and/or apsychiatric evaluation.

The term “therapy” refers to “treating” or “treatment” of a disease orcondition including preventing the disease or condition from occurringin an animal that may be predisposed to the disease but does not yetexperience or exhibit symptoms of the disease (prophylactic treatment),inhibiting the disease (slowing or arresting its development), providingrelief from the symptoms or side-effects of the disease (includingpalliative treatment), and relieving the disease (causing regression ofthe disease).

The term “effective amount,” as used herein, refers to an amount thatproduces therapeutic effects for which a substance is administered. Theeffects include the prevention, correction, or inhibition of progressionof the symptoms of a disease/condition and related complications to anydetectable extent. The exact amount will depend on the purpose of thetreatment, and will be ascertainable by one skilled in the art usingknown techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms(vols. 1-3, 1992); Lloyd, The Art, Science and Technology ofPharmaceutical Compounding (1999); and Pickar, Dosage Calculations(1999)).

The term “isolated” refers to a material that is substantially oressentially free from components, which are used to produce thematerial. For peptide conjugates of the invention, the term “isolated”refers to material that is substantially or essentially free fromcomponents, which normally accompany the material in the mixture used toprepare the peptide conjugate. “Isolated” and “pure” are usedinterchangeably. Typically, isolated peptide conjugates of the inventionhave a level of purity preferably expressed as a range. The lower end ofthe range of purity for the peptide conjugates is about 60%, about 70%or about 80% and the upper end of the range of purity is about 70%,about 80%, about 90% or more than about 90%.

When the peptide conjugates are more than about 90% pure, their puritiesare also preferably expressed as a range. The lower end of the range ofpurity is about 90%, about 92%, about 94%, about 96% or about 98%. Theupper end of the range of purity is about 92%, about 94%, about 96%,about 98% or about 100% purity.

Purity is determined by any art-recognized method of analysis (e.g.,band intensity on a silver stained gel, polyacrylamide gelelectrophoresis, HPLC, or a similar means).

“Essentially each member of the population,” as used herein, describes acharacteristic of a population of peptide conjugates of the invention inwhich a selected percentage of the modified sugars added to a peptideare added to multiple, identical acceptor sites on the peptide.“Essentially each member of the population” speaks to the “homogeneity”of the sites on the peptide conjugated to a modified sugar and refers toconjugates of the invention, which are at least about 80%, preferably atleast about 90% and more preferably at least about 95% homogenous.

“Homogeneity,” refers to the structural consistency across a populationof acceptor moieties to which the modified sugars are conjugated. Thus,in a peptide conjugate of the invention in which each modified sugarmoiety is conjugated to an acceptor site having the same structure asthe acceptor site to which every other modified sugar is conjugated, thepeptide conjugate is said to be about 100% homogeneous. Homogeneity istypically expressed as a range. The lower end of the range ofhomogeneity for the peptide conjugates is about 60%, about 70% or about80% and the upper end of the range of purity is about 70%, about 80%,about 90% or more than about 90%.

When the peptide conjugates are more than or equal to about 90%homogeneous, their homogeneity is also preferably expressed as a range.The lower end of the range of homogeneity is about 90%, about 92%, about94%, about 96% or about 98%. The upper end of the range of purity isabout 92%, about 94%, about 96%, about 98% or about 100% homogeneity.The purity of the peptide conjugates is typically determined by one ormore methods known to those of skill in the art, e.g., liquidchromatography-mass spectrometry (LC-MS), matrix assisted laserdesorption mass time of flight spectrometry (MALDITOF), capillaryelectrophoresis, and the like.

“Substantially uniform glycoform” or a “substantially uniformglycosylation pattern,” when referring to a glycopeptide species, refersto the percentage of acceptor moieties that are glycosylated by theglycosyltransferase of interest (e.g., fucosyltransferase). For example,in the case of a α1,2 fucosyltransferase, a substantially uniformfucosylation pattern exists if substantially all of the Galβ1,4-GlcNAc-Rand sialylated analogues thereof are fucosylated in a peptide conjugateof the invention. In the fucosylated structures set forth herein, theFuc-GlcNAc linkage is generally α6 or α3, with α6 generallypreferred. Itwill be understood by one of skill in the art, that the startingmaterial may contain glycosylated acceptor moieties (e.g., fucosylatedGalβ1,4-GlcNAc-R moieties). Thus, the calculated percent glycosylationwill include acceptor moieties that are glycosylated by the methods ofthe invention, as well as those acceptor moieties already glycosylatedin the starting material.

The term “substantially” in the above definitions of “substantiallyuniform” generally means at least about 40%, at least about 70%, atleast about 80%, or more preferably at least about 90%, and still morepreferably at least about 95% of the acceptor moieties for a particularglycosyltransferase are glycosylated.

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, they equally encompass thechemically identical substituents, which would result from writing thestructure from right to left, e.g., —CH₂O— is intended to also recite—OCH₂—.

The term “alkyl,” by itself or as part of another substituent means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include di- and multivalentradicals, having the number of carbon atoms designated (i.e. C₁-C₁₀means one to ten carbons). Examples of saturated hydrocarbon radicalsinclude, but are not limited to, groups such as methyl, ethyl, n-propyl,isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl,(cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, forexample, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Anunsaturated alkyl group is one having one or more double bonds or triplebonds. Examples of unsaturated alkyl groups include, but are not limitedto, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl,3-butynyl, and the higher homologs and isomers. The term “alkyl,” unlessotherwise noted, is also meant to include those derivatives of alkyldefined in more detail below, such as “heteroalkyl.” Alkyl groups thatare limited to hydrocarbon groups are termed “homoalkyl”.

The term “alkylene” by itself or as part of another substituent means adivalent radical derived from an alkane, as exemplified, but notlimited, by —CH₂CH₂CH₂CH₂—, and further includes those groups describedbelow as “heteroalkylene.” Typically, an alkyl (or alkylene) group willhave from 1 to 24 carbon atoms, with those groups having 10 or fewercarbon atoms being preferred in the present invention. A “lower alkyl”or “lower alkylene” is a shorter chain alkyl or alkylene group,generally having eight or fewer carbon atoms.

The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) areused in their conventional sense, and refer to those alkyl groupsattached to the remainder of the molecule via an oxygen atom, an aminogroup, or a sulfur atom, respectively.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon radical, or combinations thereof, consisting of thestated number of carbon atoms and at least one heteroatom selected fromthe group consisting of O, N, Si and S, and wherein the nitrogen andsulfur atoms may optionally be oxidized and the nitrogen heteroatom mayoptionally be quaternized. The heteroatom(s) O, N and S and Si may beplaced at any interior position of the heteroalkyl group or at theposition at which the alkyl group is attached to the remainder of themolecule. Examples include, but are not limited to, —CH₂—CH₂—O—CH₃,—CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂,—S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH3)₃, —CH₂—CH═N—OCH₃,and —CH═CH—N(CH₃)—CH₃. Up to two heteroatoms may be consecutive, suchas, for example, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH3)₃. Similarly, the term“heteroalkylene” by itself or as part of another substituent means adivalent radical derived from heteroalkyl, as exemplified, but notlimited by, —CH₂—CH₂—S—CH₂—CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. Forheteroalkylene groups, heteroatoms can also occupy either or both of thechain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino,alkylenediamino, and the like). Still further, for alkylene andheteroalkylene linking groups, no orientation of the linking group isimplied by the direction in which the formula of the linking group iswritten. For example, the formula —C(O)₂R′— represents both —C(O)₂R′—and —R′C(O)₂—.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like.

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl,” aremeant to include monohaloalkyl and polyhaloalkyl. For example, the term“halo(C₁-C₄)alkyl” is mean to include, but not be limited to,trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, andthe like.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, substituent that can be a single ring or multiple rings(preferably from 1 to 3 rings), which are fused together or linkedcovalently. The term “heteroaryl” refers to aryl groups (or rings) thatcontain from one to four heteroatoms selected from N, O, and S, whereinthe nitrogen and sulfur atoms are optionally oxidized, and the nitrogenatom(s) are optionally quaternized. A heteroaryl group can be attachedto the remainder of the molecule through a heteroatom. Non-limitingexamples of aryl and heteroaryl groups include phenyl, 1-naphthyl,2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl,2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, tetrazolyl, benzo[b]furanyl, benzo[b]thienyl,2,3-dihydrobenzo[1,4]dioxin-6-yl, benzo[1,3]dioxol-5-yl and 6-quinolyl.Substituents for each of the above noted aryl and heteroaryl ringsystems are selected from the group of acceptable substituents describedbelow.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the term “arylalkyl” is meant to includethose radicals in which an aryl group is attached to an alkyl group(e.g., benzyl, phenethyl, pyridylmethyl and the like) including thosealkyl groups in which a carbon atom (e.g., a methylene group) has beenreplaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl” and“heteroaryl”) is meant to include both substituted and unsubstitutedforms of the indicated radical. Preferred substituents for each type ofradical are provided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) are generically referred to as “alkyl groupsubstituents,” and they can be one or more of a variety of groupsselected from, but not limited to: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂ in a number ranging from zero to (2m′+1), where m′ is the totalnumber of carbon atoms in such radical. R′, R″, R′″ and R″″ eachpreferably independently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, e.g., aryl substitutedwith 1-3 halogens, substituted or unsubstituted alkyl, alkoxy orthioalkoxy groups, or arylalkyl groups. When a compound of the inventionincludes more than one R group, for example, each of the R groups isindependently selected as are each R′, R″, R′″ and R″″ groups when morethan one of these groups is present. When R′ and R″ are attached to thesame nitrogen atom, they can be combined with the nitrogen atom to forma 5-, 6-, or 7-membered ring. For example, —NR′R″ is meant to include,but not be limited to, 1-pyrrolidinyl and 4-morpholinyl. From the abovediscussion of substituents, one of skill in the art will understand thatthe term “alkyl” is meant to include groups including carbon atoms boundto groups other than hydrogen groups, such as haloalkyl (e.g., —CF₃ and—CH₂CF₃) and acyl (e.g., —C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and thelike).

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are generically referredto as “aryl group substituents.” The substituents are selected from, forexample: halogen, —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′, -halogen,—SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy, and fluoro(C₁-C₄)alkyl,in a number ranging from zero to the total number of open valences onthe aromatic ring system; and where R′, R″, R′″ and R″″ are preferablyindependently selected from hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted aryl and substituted or unsubstituted heteroaryl. When acompound of the invention includes more than one R group, for example,each of the R groups is independently selected as are each R′, R″, R′″and R″″ groups when more than one of these groups is present. In theschemes that follow, the symbol X represents “R” as described above.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally be replaced with a substituent of the formula-T-C(O)—(CRR′)q-U-, wherein T and U are independently —NR—, —O—, —CRR′—or a single bond, and q is an integer of from 0 to 3. Alternatively, twoof the substituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula -A-(CH₂)r-B—,wherein A and B are independently —CRR′—, —O—, —NR—, —S—, —S(O)—,—S(O)₂—, —S(O)₂NR′— or a single bond, and r is an integer of from 1 to4. One of the single bonds of the new ring so formed may optionally bereplaced with a double bond. Alternatively, two of the substituents onadjacent atoms of the aryl or heteroaryl ring may optionally be replacedwith a substituent of the formula —(CRR′)s-X—(CR″R′″)d-, where s and dare independently integers of from 0 to 3, and X is —O—, —NR′—, —S—,—S(O)—, —S(O)₂—, or —S(O)₂NR′—. The substituents R, R′, R″ and R′″ arepreferably independently selected from hydrogen or substituted orunsubstituted (C1-C6)alkyl.

As used herein, the term “heteroatom” is meant to include oxygen (O),nitrogen (N), sulfur (S) and silicon (Si).

Introduction

To improve the effectiveness of recombinant human growth hormone (rhGH)used for therapeutic purposes, the present invention providesgenetically engineered mutants of human growth hormone that containN-linked and/or O-linked glycosylation sites not present in naturallyoccurring human growth hormone. To further minimize in vivo degradationand lower the clearance rate of recombinant human growth hormone in thebody, some of the mutants contain a proteolysis resistant mutation(s)and/or potential sites for chemical PEGylation in regions susceptible toproteases. FIGS. 6A-6K illustrate exemplary proteolysis resistantmutation sites. While hGH mutants of the invention also substantiallyretain the biological activity of the wild-type hormone, the newlyintroduced glycosylation sites allow the recombinantly produced hGHmutants to be selectively glycosylated in a large variety of patterns.Moreover, the non-natural glycosylation sites provide loci for selectiveglycoconjugation of modifying groups to the peptide at one or moresites. An exemplary modifying group is a water-soluble polymer, such aspoly(ethylene glycol), e.g., PEG (e.g., m-PEG), PPG (e.g., m-PPG).Modification of the hGH mutants improve the stability and in vivoretention time of the recombinant hGH, reduces the peptides'antigenicity, and enhances the peptides' ability to target a specifictissue in need of treatment.

The Mutants

Glycosylation Mutants

The present invention provides mutants of hGH that include one or moreO- or N-linked glycosylation sites that are not found in the wild typepeptide. In all cases, the N-terminal Met may be present or absent onany hGH mutant. The mutants are substrates for enzymatic glycosylationand/or glycoPEGylation at one or more sites that would not normally beglycosylated, or would be poorly glycosylated, in the wild type peptide.These mutants allow the position of a glycosyl residue or a glycosyllinking group to be engineered to obtain a peptide having selecteddesirable properties. In addition to the position and number of glycosylresidues or glycosyl linking groups, other properties that can be variedusing the mutants and methods of the invention include pharmacokinetics,pharmacodynamics, resistance to proteolysis, immunogenicity, recognitionby the reticuloendothelial system, tissue distribution and the like.

Exemplary glycosylation mutants include the following: P134TTGQIF= Mutant BD:MFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPTTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF P134TTAQIF = MutantBE:MFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPTTAQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF P134TATQIF = MutantBF:MFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPTATQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF P134TQGAMF = MutantAI:MFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPTQGAMFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF P134TQGAIF = Mutant BG:MFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPTQGAIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF P134TQGQIF = MutantBH:MFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPTQGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF P134TTLYVF = MutantBI:MFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPTTLYVFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF P134TINTIF = MutantAJ:MFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPTINTIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF P134TTVSIF = Mutant AH:MFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPTTVSIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF I139PTQTYS = MutantBJ:MFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIPTQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF I139PTQAYS = MutantAG:MFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIPTQAYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF P134TTLQIF = Mutant BK:MFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPTTLQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF P134TTVQIF = MutantBL:MFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPTTVQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF P134TTNQIF = MutantBM:MFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPTTNQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF P134TTQQIF = MutantBN:MFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPTTQQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF P134TIGQIF = MutantBO:MFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPTIGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF P134TILQIF = MutantBP:MFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPTILQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF P134TIVQIF = MutantBQ:MFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPTIVQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF P134TINQIF = MutantBR:MFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPTINQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF P134TIQQIF = MutantBS:MFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPTIQQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF P134TIAQIF = MutantBT:MFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPTIAQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF L129ETETPRT= Mutant BU:MFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLETETPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF L129VTETPRT= Mutant BV:MFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLVTETPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF L129ETQSPRT= Mutant BW:MFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLETQSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF L129VTQSPRT= Mutant BX:MFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLVTQSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF L129VTETPAT= Mutant BY:MFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLVTETPATGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF L129ETETPAT= Mutant BZ:MFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLETETPATGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF L129ATGSPRT= Mutant CA:MFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLATGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF L129ETQSPST= Mutant CB:MFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLETQSPSTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF L129ETQSPAT= Mutant CC:MFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLETQSPATGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF L129ETQSPLT= Mutant CD:MFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLETQSPLTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF Y43change to A;P134TINTIFKQTYS = Mutant CE:MFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKASFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPTINTIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF Y43 change to A; Y144 change to A;P134TINTIFKQTA = Mutant CF:MFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKASFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPTINTIFKQTASKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF Y43 change to A; Y144 change to A;F140 change to A; K141 change to N; P134TINTIANQTA = Mutant CG:MFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKASFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPTINTIANQTASKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF Y43 change to A; I139PTQAYS= Mutant CH:MFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKASFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIPTQAYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF V1TPT = Mutant CI:VTPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF P134TQGAMP = Mutant CJ:MFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPTQGAMPKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF P134TTTQIF = Mutant CK:MFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPTTTQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF P134NTGQIF = Mutant CL:MFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPNTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF M1FPTEIP = Mutant CM:MFPTEIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLGFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF M1FPTVLP = Mutant CN:MFPTVLPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGFA. GlycoPEGylated rhGH Mutants

The present invention provides glycoPEGylated mutants of hGH thatinclude one or more O- or N-linked glycosylation sites that are notfound in the wild type peptide. In all cases, the N-terminal Met may bepresent or absent on any hGH mutant. In addition to the position andnumber of glycosyl residues or glycosyl linking groups, other propertiesthat can be varied using the mutants and methods of the inventioninclude pharmacokinetics, pharmacodynamics, resistance to proteolysis,immunogenicity, recognition by the reticuloendothelial system, tissuedistribution and the like.

B. Proteolysis Resistant Mutants

The present invention also provides mutants of hGH with mutations thatimpart resistance towards degradation of the peptide by proteolyticenzymes. In all cases, the N-terminal Met may be present or absent onany hGH mutant. Protease resistance is achieved by modifying,substituting, inserting or deleting one or more amino acids within theprotease recognition or cleavage sites in rhGH. Exemplary mutant motifsinclude, but is not limited to the following:

wherein horizontal lines on both sides of the various motifs representamino acid sequences which precede and follow it in the correspondingnative hGH amino acid sequence. For purpose of illustration, examples ofthe P¹³⁴TINT motif have been provided in FIGS. 6D, 6E, 6F, 6G, and 6J.In each of these five mutants, the native R¹³⁵TGQ sequence has beenreplaced by TINT.

Examples of the P¹⁴⁰TQA motif are provided in FIGS. 6C and 6H, andexamples of the P¹³⁴TTVS motif in FIGS. 6B and 6I. In FIGS. 6C and 6H,the native FKQT motif has been replaced by PTQA. In FIGS. 6B and 6I, thenative R¹³⁵TGQ sequence has been replaced by T¹³⁵TVS. An exemplaryinsertion mutant can be found in FIG. 6A, where a glutamic acid has beeninserted between the threonine at the 4th position and isoleucine at the5th position of the native hGH sequence.

In addition to what is provided in FIGS. 6A to 6J, FIG. 6K shows otherpossible sites for the proteolysis resistant mutations, as indicated onthe native hGH amino acid sequence. Protease resistant mutations can bethe sole mutation type or the mutant peptide can also include one ormore glycosylation mutation to form a protease resistant conjugate thatcan also serve as substrates for enzymatic glycosylation and/orglycoPEGylation. FIGS. 6A-6J depict various exemplary mutant hGH aminoacid sequences containing both a glycosylation site and at least onetype of the proteolysis inhibiting mutations mentioned above.

C. Chemically PEGylated rhGH

The present invention further provides wild-type hGH peptides and hGHmutants that are substrates for chemical PEGylation.

Protease resistance can also be achieved through a mechanism other thanthe proteolysis resistant mutations described above. Since many of thelysine residues in hGH are present in regions most susceptible toproteases, chemical PEGylation of one or more of these lysines has theeffect of blocking the proteolytic cleavage or binding site, whichthereby reduces in vivo degradation of the polypeptide. The aminoterminus and histidine residues in hGH are other useful sites forchemical PEGylation. Exemplary chemical PEGylation sites of theinvention are indicated by larger, highlighted, bold font in FIG. 6I andFIG. 6J.

Mixed Modalities

While chemical PEGylation can be used alone, combining glycoPEGylationwith chemical PEGylation or protease resistant mutation(s) creates aprotein superior to what is known in the art. These mutants have thedual advantage of extended serum/blood half-life and increasedresistance to proteolysis. Extended residence time in the body isachieved through the introduction of a PEG at the glycoPEGylation sitewhereas reduced susceptibility to proteolysis is promoted either throughchemical PEGylation or a protease resistant mutation. Exemplary aminoacid sequences of mutants that can be employed in this twofold approachare shown in FIGS. 6I and 6J. For illustrative purposes, the methionineresidue at the N-terminus and lysine residues have been highlighted toshow exemplary sites for chemical PEGylation.

The present invention also provides for chemically PEGylated hGH mutantscontaining protease resistant mutations. In these conjugates, thecombined use of chemical PEGylation of residues near or in the proteasecleavage/recognition site and protease resistant mutations provides atwo-pronged method of blocking proteolytic activity. In addition,proteolysis inhibiting mutations can be selectively introduced so as toprovide amino-containing side chains, which can be used as additionalsubstrates for chemical PEGylation. exemplary sites for chemicalPEGylation.

Moreover, chemical PEGylation, glycoPEGylation, and proteolysisinhibiting mutations can all be used in conjunction, as exemplified byFIGS. 6A-6K. Each of these amino acid sequences contains proteolysisinhibiting mutations and glycosylation sites according to the presentinvention. Preferably, though not necessary to practice this aspect ofthe invention, glycosylation site mutations and proteolysis inhibitingmutations are introduced in such a manner as to retain at least onelysine or histidine residue within the mutant amino acid sequence.

The present invention provides for hGH mutants with N-linked or O-linkedglycosylation sites not found in wild-type hGH. Exemplary embodiments ofthe invention include N- or O-linked glycosylated hGH mutants having oneor more characteristics selected from the following: glycoPEGylation,protease resistance, and chemically PEGylation. Through the controlledmodification of hGH, the present invention yields novel hGH derivativeswith pharmacokinetic properties that are improved relative to thecorresponding native hGH.

In a first aspect, the present invention provides an isolated nucleicacid comprising a polynucleotide sequence encoding a mutant human growthhormone. The mutant human growth hormone comprises an N-linked orO-linked glycosylation site and/or proteolysis resistant mutation(s)that are not present in wild-type human growth hormone. In exemplaryembodiments, the wild-type human growth hormones have the amino acidsequence of pituitary-derived GH-N (SEQ ID NO:1) or placenta-derivedGH-V (SEQ ID NO:2). In some preferred embodiments, the mutant humangrowth hormone includes the amino acid sequence of SEQ ID NO:3, 4, 5, 6,7, 8, 9, 80, 81, 82, 83, 84, 85, 86, 87, and 88.

In another aspect, the present invention provides an expression cassetteor a cell that comprises a nucleic acid, e.g., an isolated nucleic acid,including a polynucleotide sequence encoding a mutant human growthhormone. The mutant human growth hormone includes an N-linked orO-linked glycosylation site and/or proteolysis inhibiting mutation(s)that are not present in the wild-type human growth hormone.

In still another aspect, the present invention provides a mutantglycoPEGylated human growth hormone that includes one or more N-linkedor O-linked glycosylation site not present in the wild-type human growthhormone. In some embodiments, the wild-type human growth hormone has theamino acid sequence of SEQ ID NO:1 or SEQ ID NO:2. In some preferredembodiments, the mutant human growth hormone comprises the amino acidsequence of SEQ ID NO:3, 4, 5, 6, 7, 8, 9, 80, 81, 82, 83, 84, 85, 86,87, and 88.

In still another aspect, the present invention provides a mutant humangrowth hormone that includes one or more N-linked or O-linkedglycosylation site not present in the wild-type human growth hormone andone or more proteolysis resistant mutations(s) not present in thewild-type human growth hormone. In some embodiments, the wild-type humangrowth hormone has the amino acid sequence of SEQ ID NO:1 or SEQ IDNO:2. In some preferred embodiments, the mutant human growth hormonecomprises the amino acid sequence of SEQ ID NO:3, 4, 5, 6, 7, 8, 9, 80,81, 82, 83, 84, 85, 86, 87, and 88.

In still another aspect, the present invention provides a chemicallyPEGylated mutant human growth hormone that includes one or more N-linkedor O-linked glycosylation site not present in the wild-type human growthhormone. In some embodiments, the wild-type human growth hormone has theamino acid sequence of SEQ ID NO:1 or SEQ BD NO:2. In some preferredembodiments, the mutant human growth hormone comprises the amino acidsequence of SEQ ID NO:3, 4, 5, 6, 7, 8, 9, 80, 81, 82, 83, 84, 85, 86,87, and 88.

In another aspect, the present invention provides a glycoPEGylatedmutant human growth hormone that includes one or more N-linked orO-linked glycosylation site not present in the wild-type human growthhormone and one or more proteolysis resistant mutations(s) not presentin the wild-type human growth hormone. In some embodiments, thewild-type human growth hormone has the amino acid sequence of SEQ IDNO:1 or SEQ ID NO:2. In some preferred embodiments, the mutant humangrowth hormone comprises the amino acid sequence of SEQ ID NO:3, 4, 5,6, 7, 8, 9, 80, 81, 82, 83, 84, 85, 86, 87, and 88.

In another aspect, the present invention provides a chemically PEGylatedmutant human growth hormone that includes one or more N-linked orO-linked glycosylation site not present in the wild-type human growthhormone and one or more proteolysis resistant mutations(s) not presentin the wild-type human growth hormone. In some embodiments, thewild-type human growth hormone has the amino acid sequence of SEQ IDNO:1 or SEQ ID NO:2. In some preferred embodiments, the mutant humangrowth hormone comprises the amino acid sequence of SEQ ID NO:3, 4, 5,6, 7, 8, 9, 80, 81, 82, 83, 84, 85, 86, 87, and 88.

In another aspect, the present invention provides a glycoPEGylated andchemically PEGylated mutant human growth hormone that includes one ormore N-linked or O-linked glycosylation site not present in thewild-type human growth hormone. In some embodiments, the wild-type humangrowth hormone has the amino acid sequence of SEQ ID NO:1 or SEQ IDNO:2. In some preferred embodiments, the mutant human growth hormonecomprises the amino acid sequence of SEQ ID NO:3, 4, 5, 6, 7, 8, 9, 80,81, 82, 83, 84, 85, 86, 87, and 88.

In another aspect, the present invention provides a glycoPEGylated andchemically PEGylated mutant human growth hormone that includes one ormore N-linked or O-linked glycosylation site not present in thewild-type human growth hormone and one or more proteolysis resistantmutations(s) not present in the wild-type human growth hormone. In someembodiments, the wild-type human growth hormone has the amino acidsequence of SEQ ID NO:1 or SEQ ID NO:2. In some preferred embodiments,the mutant human growth hormone comprises the amino acid sequence of SEQID NO:3, 4, 5, 6, 7, 8, 9, 80, 81, 82, 83, 84, 85, 86, 87, and 88.

In another aspect, the present invention provides a method for making amutant human growth hormone that includes an N-linked or O-linkedglycosylation site and/or proteolysis inhibiting mutation(s) that arenot present in the wild-type human growth hormone. This method includesthe steps of recombinantly producing the mutant human growth hormone,and glycosylating the mutant human growth hormone at the newglycosylation site. In some embodiments, the wild-type human growthhormone has the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2. Insome preferred embodiments, the mutant human growth hormone comprisesthe amino acid sequence of SEQ ID NO:3, 4, 5, 6, 7, 8, 9, 80, 81, 82,83, 84, 85, 86, 87, and 88.

In still a further aspect, the present invention provides apharmaceutical composition having a therapeutically effective amount ofa mutant human growth hormone that includes an N-linked or O-linkedglycosylation site and/or proteolysis inhibiting mutation(s) not presentin the wild-type human growth hormone. In some embodiments, thewild-type human growth hormone has the amino acid sequence of SEQ IDNO:1 or SEQ ID NO:2. In some preferred embodiments, the mutant humangrowth hormone comprises the amino acid sequence of SEQ ID NO:3, 4, 5,6, 7, 8, 9, 80, 81, 82, 83, 84, 85, 86, 87, and 88.

In another aspect, the present invention provides a method for treatinghuman growth hormone deficiency in a subject. The method includesadministering to the subject an amount of a mutant human growth hormoneeffective to treat or ameliorate the growth hormone deficiency. Themutant human growth hormone used in this method comprises an N-linked orO-linked glycosylation site and/or proteolysis inhibiting mutation(s)that do not exist in the corresponding wild-type human growth hormone.In some embodiments, the corresponding wild-type human growth hormonehas the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2. In somepreferred embodiments, the mutant human growth hormone comprises theamino acid sequence of SEQ ID NO:3, 4, 5, 6, 7, 8, 9, 80, 81, 82, 83,84, 85, 86, 87, and 88.

Acquisition of hGH Coding Sequences

General Recombinant Technology

This invention relies on routine techniques in the field of recombinantgenetics. Basic texts disclosing the general methods of use in thisinvention include Sambrook and Russell, Molecular Cloning, A LaboratoryManual (3rd ed. 2001); Kriegler, Gene Transfer and Expression: ALaboratory Manual (1990); and Ausubel et al., eds., Current Protocols inMolecular Biology (1994).

A number of polynucleotide sequences encoding a wild-type human growthhormone, e.g., GenBank Accession Nos. NM 000515, NM 002059, NM 022556,NM 022557, NM 022558, NM 022559, NM 022560, NM 022561, and NM 022562,have been determined and can be obtained from a commercial supplier.

Introducing Mutations into an hGH Sequence

From an encoding polynucleotide sequence, the amino acid sequence of awild-type human growth hormone, e.g., SEQ ID NO:1 or SEQ ID NO:2, can bedetermined. Subsequently, this amino acid sequence may be modified tointroduce proteolysis inhibiting mutations and/or alter the protein'sglycosylation pattern, by introducing additional glycosylation site(s)at various locations in the amino acid sequence.

Several types of protein glycosylation sites are well known in the art.For instance, in eukaryotes, N-linked glycosylation occurs on theasparagine of the consensus sequence Asn-X_(aa)-Ser/Thr, in which X_(aa)is any amino acid except proline (Kornfeld et al., Ann Rev Biochem54:631-664 (1985); Kukuruzinska et al., Proc. Natl. Acad. Sci. USA84:2145-2149 (1987); Herscovics et al., FASEB J 7:540-550 (1993); andOrlean, Saccharomyces Vol. 3 (1996)). O-linked glycosylation takes placeat serine or threonine residues (Tanner et al., Biochim. Biophys. Acta.906:81-91 (1987); and Hounsell et al., Glycoconj. J. 13:19-26 (1996)).Other glycosylation patterns are formed by linkingglycosylphosphatidylinositol to the carboxyl-terminal carboxyl group ofthe protein (Takeda et al., Trends Biochem. Sci. 20:367-371 (1995); andUdenfriend et al., Ann. Rev. Biochem. 64:593-591 (1995). Based on thisknowledge, suitable mutations can thus be introduced into a wild-typehuman growth hormone sequence to form new glycosylation sites.

Although direct modification of an amino acid residue within a humangrowth hormone polypeptide sequence may be feasible to introduce a newN-linked or O-linked glycosylation site, more frequently, introductionof a new glycosylation site is accomplished by mutating thepolynucleotide sequence encoding a human growth hormone. This can beachieved by using any of known mutagenesis methods, some of which arediscussed below. Exemplary modifications to human growth hormone includethose illustrated in SEQ ID NOS: 3-9, and 80-89.

A variety of mutation-generating protocols are established and describedin the art. See, e.g., Zhang et al., Proc. Natl. Acad. Sci. USA, 94:4504-4509 (1997); and Stemmer, Nature, 370: 389-391 (1994). Theprocedures can be used separately or in combination to produce variantsof a set of nucleic acids, and hence variants of encoded polypeptides.Kits for mutagenesis, library construction, and otherdiversity-generating methods are commercially available.

Mutational methods of generating diversity include, for example,site-directed mutagenesis (Botstein and Shortle, Science, 229: 1193-1201(1985)), mutagenesis using uracil-containing templates (Kunkel, Proc.Natl. Acad. Sci. USA, 82: 488-492 (1985)), oligonucleotide-directedmutagenesis (Zoller and Smith, Nucl. Acids Res., 10: 6487-6500 (1982)),phosphorothioate-modified DNA mutagenesis (Taylor et al., Nucl. AcidsRes., 13: 8749-8764 and 8765-8787 (1985)), and mutagenesis using gappedduplex DNA (Kramer et al., Nucl. Acids Res., 12: 9441-9456 (1984)).

Other possible methods for generating mutations include point mismatchrepair (Kramer et al., Cell, 38: 879-887 (1984)), mutagenesis usingrepair-deficient host strains (Carter et al., Nucl. Acids Res., 13:4431-4443 (1985)), deletion mutagenesis (Eghtedarzadeh and Henikoff,Nucl. Acids Res., 14: 5115 (1986)), restriction-selection andrestriction-purification (Wells et al., Phil. Trans. R. Soc. Lond. A,317: 415-423 (1986)), mutagenesis by total gene synthesis (Nambiar etal., Science, 223: 1299-1301 (1984)), double-strand break repair(Mandecki, Proc. Natl. Acad. Sci. USA, 83: 7177-7181 (1986)),mutagenesis by polynucleotide chain termination methods (U.S. Pat. No.5,965,408), and error-prone PCR (Leung et al., Biotechniques, 1: 11-15(1989)).

Following sequence verification, the mutant human growth hormone of thepresent invention can be produced using routine techniques in the fieldof recombinant genetics, relying on the polynucleotide sequencesencoding the polypeptide disclosed herein.

To obtain high level expression of a nucleic acid encoding a mutanthuman growth hormone of the present invention, one typically subclones apolynucleotide encoding the mutant human growth hormone into anexpression vector that contains a strong promoter to directtranscription, a transcription/translation terminator and a ribosomebinding site for translational initiation. Suitable bacterial promotersare well known in the art and described, e.g., in Sambrook and Russell,supra, and Ausubel et al., supra. Bacterial expression systems forexpressing the wild-type or mutant human growth hormone are availablein, e.g., E. coli, Bacillus sp., Salmonella, and Caulobacter. Kits forsuch expression systems are commercially available. Eukaryoticexpression systems for mammalian cells, yeast, and insect cells are wellknown in the art and are also commercially available.

When periplasmic expression of a recombinant protein (e.g., a hGH mutantof the present invention) is desired, the expression vector furthercomprises a sequence encoding a secretion signal, such as the E. coliOppA (Periplasmic Oligopeptide Binding Protein) secretion signal or amodified version thereof, which is directly connected to 5′ of thecoding sequence of the protein to be expressed. This signal sequencedirects the recombinant protein produced in cytoplasm through the cellmembrane into the periplasmic space. The expression vector may furthercomprise a coding sequence for signal peptidase 1, which is capable ofenzymatically cleaving the signal sequence when the recombinant proteinis entering the periplasmic space. More detailed description forperiplasmic production of a recombinant protein can be found in, e.g.,Gray et al., Gene 39: 247-254 (1985), U.S. Pat. Nos. 6,160,089 and6,436,674.

As discussed above, a person skilled in the art will recognize thatvarious conservative substitutions can be made to any wild-type ormutant human growth hormone or its coding sequence while still retainingthe biological activity of the human growth hormone. Moreover,modifications of a polynucleotide coding sequence may also be made toaccommodate preferred codon usage in a particular expression hostwithout altering the resulting amino acid sequence.

Standard transfection methods are used to produce bacterial, mammalian,yeast, insect, or plant cell lines that express large quantities of themutant human growth hormone, which are then purified using standardtechniques (see, e.g., Colley et al., J. Biol. Chem. 264: 17619-17622(1989); Guide to Protein Purification, in Methods in Enzymology, vol.182 (Deutscher, ed., 1990)). Transformation of eukaryotic andprokaryotic cells are performed according to standard techniques (see,e.g., Morrison, J. Bact. 132: 349-351 (1977); Clark-Curtiss & Curtiss,Methods in Enzymology 101: 347-362 (Wu et al., eds, 1983).

Any of the well known procedures for introducing foreign nucleotidesequences into host cells may be used. These include the use of calciumphosphate transfection, polybrene, protoplast fusion, electroporation,liposomes, microinjection, plasma vectors, viral vectors and any of theother well known methods for introducing cloned genomic DNA, cDNA,synthetic DNA, or other foreign genetic material into a host cell (see,e.g., Sambrook and Russell, supra). It is only necessary that theparticular genetic engineering procedure used be capable of successfullyintroducing at least one gene into the host cell capable of expressingthe mutant human growth hormone.

After the expression vector is introduced into appropriate host cells,the transfected cells are cultured under conditions favoring expressionof the mutant human growth hormone. The cells are then screened for theexpression of the recombinant polypeptide, which is subsequentlyrecovered from the culture using standard techniques (see, e.g., Scopes,Protein Purification: Principles and Practice (1982); U.S. Pat. No.4,673,641; Ausubel et al., supra; and Sambrook and Russell, supra).

Several general methods for screening gene expression are well knownamong those skilled in the art. First, gene expression can be detectedat the nucleic acid level. A variety of methods of specific DNA and RNAmeasurement using nucleic acid hybridization techniques are commonlyused (e.g., Sambrook and Russell, supra). Some methods involve anelectrophoretic separation (e.g., Southern blot for detecting DNA andNorthern blot for detecting RNA), but detection of DNA or RNA can becarried out without electrophoresis as well (such as by dot blot). Thepresence of nucleic acid encoding a mutant human growth hormone intransfected cells can also be detected by PCR or RT-PCR usingsequence-specific primers.

Second, gene expression can be detected at the polypeptide level.Various immunological assays are routinely used by those skilled in theart to measure the level of a gene product, particularly usingpolyclonal or monoclonal antibodies that react specifically with amutant human growth hormone of the present invention, such as apolypeptide having the amino acid sequence of SEQ ID NO:3, 4, or 5,(e.g., Harlow and Lane, Antibodies, A Laboratory Manual, Chapter 14,Cold Spring Harbor, 1988; Kohler and Milstein, Nature, 256: 495-497(1975)). Such techniques require antibody preparation by selectingantibodies with high specificity against the mutant human growth hormoneor an antigenic portion thereof. The methods of raising polyclonal andmonoclonal antibodies are well established and their descriptions can befound in the literature, see, e.g., Harlow and Lane, supra; Kohler andMilstein, Eur. J. Immunol., 6: 511-519 (1976). More detaileddescriptions of preparing antibody against the mutant human growthhormone of the present invention and conducting immunological assaysdetecting the mutant human growth hormone are provided in a latersection.

Purification of Recombinantly Produced Mutant hGH

In one exemplary embodiment, art-recognized methods for purifyingbacterially-expressed peptides are utilized. For example, when themutant human growth hormones of the present invention are producedrecombinantly by transformed bacteria in large amounts, typically afterpromoter induction, although expression can be constitutive, theproteins may form insoluble aggregates. There are several protocols thatare suitable for purification of protein inclusion bodies. Purificationof aggregate proteins (hereinafter referred to as inclusion bodies)typically involves the extraction, separation and/or purification ofinclusion bodies by disruption of bacterial cells, e.g., by incubationin a buffer of about 100-150 μg/ml lysozyme and 0.1% Nonidet P40, anon-ionic detergent. The cell suspension can be ground using a Polytrongrinder (Brinkman Instruments, Westbury, N.Y.). Alternatively, the cellscan be sonicated on ice. Alternate methods of lysing bacteria aredescribed in Ausubel et al. and Sambrook and Russell, both supra, andwill be apparent to those of skill in the art.

The cell suspension is generally centrifuged and the pellet containingthe inclusion bodies resuspended in buffer which does not dissolve butwashes the inclusion bodies, e.g., 20 mM Tris-HCl (pH 7.2), 1 mM EDTA,150 mM NaCl and 2% Triton-X 100, a non-ionic detergent. It may benecessary to repeat the wash step to remove as much cellular debris aspossible. The remaining pellet of inclusion bodies may be resuspended inan appropriate buffer (e.g., 20 mM sodium phosphate, pH 6.8, 150 mMNaCl). Other appropriate buffers will be apparent to those of skill inthe art.

Following the washing step, the inclusion bodies are solubilized by theaddition of a solvent that is both a strong hydrogen acceptor and astrong hydrogen donor (or a combination of solvents each having one ofthese properties). The proteins that formed the inclusion bodies maythen be renatured by dilution or dialysis with a compatible buffer.Suitable solvents include, but are not limited to, urea (from about 4 Mto about 8 M), formamide (at least about 80%, volume/volume basis), andguanidine hydrochloride (from about 4 M to about 8 M). Some solventsthat are capable of solubilizing aggregate-forming proteins, such as SDS(sodium dodecyl sulfate) and 70% formic acid, may be inappropriate foruse in this procedure due to the possibility of irreversibledenaturation of the proteins, accompanied by a lack of immunogenicityand/or activity. Although guanidine hydrochloride and similar agents aredenaturants, this denaturation is not irreversible and renaturation mayoccur upon removal (by dialysis, for example) or dilution of thedenaturant, allowing re-formation of the immunologically and/orbiologically active protein of interest. After solubilization, theprotein can be separated from other bacterial proteins by standardseparation techniques. For further description of purifying recombinanthuman growth hormone from bacterial inclusion body, see, e.g., Patra etal., Protein Expression and Purification 18: 182-190 (2000).

Alternatively, it is possible to purify recombinant polypeptides, e.g.,a mutant human growth hormone, from bacterial periplasm. Where therecombinant protein is exported into the periplasm of the bacteria, theperiplasmic fraction of the bacteria can be isolated by cold osmoticshock in addition to other methods known to those of skill in the art(see e.g., Ausubel et al., supra). To isolate recombinant proteins fromthe periplasm, the bacterial cells are centrifuged to form a pellet. Thepellet is resuspended in a buffer containing 20% sucrose. To lyse thecells, the bacteria are centrifuged and the pellet is resuspended inice-cold 5 mM MgSO₄ and kept in an ice bath for approximately 10minutes. The cell suspension is centrifuged and the supernatant decantedand saved. The recombinant proteins present in the supernatant can beseparated from the host proteins by standard separation techniques wellknown to those of skill in the art.

Standard Protein Separation Techniques for Purification

When a recombinant polypeptide, e.g., the mutant human growth hormone ofthe present invention, is expressed in host cells in a soluble form, itspurification can follow the standard protein purification proceduredescribed below.

Solubility Fractionation

Often as an initial step, and if the protein mixture is complex, aninitial salt fractionation can separate many of the unwanted host cellproteins (or proteins derived from the cell culture media) from therecombinant protein of interest, e.g., a mutant human growth hormone ofthe present invention. The preferred salt is ammonium sulfate. Ammoniumsulfate precipitates proteins by effectively reducing the amount ofwater in the protein mixture. Proteins then precipitate on the basis oftheir solubility. The more hydrophobic a protein is, the more likely itis to precipitate at lower ammonium sulfate concentrations. A typicalprotocol is to add saturated ammonium sulfate to a protein solution sothat the resultant ammonium sulfate concentration is between 20-30%.This will precipitate the most hydrophobic proteins. The precipitate isdiscarded (unless the protein of interest is hydrophobic) and ammoniumsulfate is added to the supernatant to a concentration known toprecipitate the protein of interest. The precipitate is then solubilizedin buffer and the excess salt removed if necessary, through eitherdialysis or diafiltration. Other methods that rely on solubility ofproteins, such as cold ethanol precipitation, are well known to those ofskill in the art and can be used to fractionate complex proteinmixtures.

Size Differential Filtration

Based on a calculated molecular weight, a protein of greater and lessersize can be isolated using ultrafiltration through membranes ofdifferent pore sizes (for example, Amicon or Millipore membranes). As afirst step, the protein mixture is ultrafiltered through a membrane witha pore size that has a lower molecular weight cut-off than the molecularweight of a protein of interest, e.g., a mutant human growth hormone.The retentate of the ultrafiltration is then ultrafiltered against amembrane with a molecular cut off greater than the molecular weight ofthe protein of interest. The recombinant protein will pass through themembrane into the filtrate. The filtrate can then be chromatographed asdescribed below.

Column Chromatography

The proteins of interest (such as the mutant human growth hormone of thepresent invention) can also be separated from other proteins on thebasis of their size, net surface charge, hydrophobicity, or affinity forligands. In addition, antibodies raised against human growth hormone canbe conjugated to column matrices and the human growth hormoneimmunopurified. All of these methods are well known in the art.

It will be apparent to one of skill that chromatographic techniques canbe performed at any scale and using equipment from many differentmanufacturers (e.g., Pharmacia Biotech).

Immunoassays for Detection of Mutant hGH Expression

To confirm the production of a recombinant mutant human growth hormone,immunological assays may be useful to detect in a sample the expressionof the polypeptide. Immunological assays are also useful for quantifyingthe expression level of the recombinant hormone. Antibodies against amutant human growth hormone are necessary for carrying out theseimmunological assays.

Methods for producing polyclonal and monoclonal antibodies that reactspecifically with an immunogen of interest are known to those of skillin the art (see, e.g., Coligan, Current Protocols in ImmunologyWiley/Greene, NY, 1991; Harlow and Lane, Antibodies: A Laboratory ManualCold Spring Harbor Press, NY, 1989; Stites et al. (eds.) Basic andClinical Immunology (4th ed.) Lange Medical Publications, Los Altos,Calif., and references cited therein; Goding, Monoclonal Antibodies:Principles and Practice (2d ed.) Academic Press, New York, N.Y., 1986;and Kohler and Milstein Nature 256: 495-497, 1975). Such techniquesinclude antibody preparation by selection of antibodies from librariesof recombinant antibodies in phage or similar vectors (see, Huse et al.,Science 246: 1275-1281, 1989; and Ward et al., Nature 341: 544-546,1989).

Immunoassays for Detecting Mutant hGH Expression

Once antibodies specific for a mutant human growth hormone of thepresent invention are available, the amount of the polypeptide in asample, e.g., a cell lysate, can be measured by a variety of immunoassaymethods providing qualitative and quantitative results to a skilledartisan. For a review of immunological and immunoassay procedures ingeneral see, e.g., Stites, supra; U.S. Pat. Nos. 4,366,241; 4,376,110;4,517,288; and 4,837,168.

Glycosylation and Glycoconjugation of the Mutant hGH

Glycosylation and Glycoconjugation by Enzymatic Methods

Post-expression in vitro modification of peptides is an attractivestrategy to remedy the deficiencies of methods that rely on controllingglycosylation by engineering expression systems, including bothmodification of glycan structures or introduction of glycans at novelsites. A comprehensive arsenal of enzymes that transfer saccharide donormoieties is becoming available, making in vitro enzymatic synthesis ofglycoconjugates with custom designed glycosylation patterns and glycosylstructures possible. See, for example, U.S. Pat. Nos. 5,876,980;6,030,815; 5,728,554; 5,922,577; and published patent applications WO98/31826; WO 01/88117; WO 03/031464; WO 03/046150; WO 03/045980; WO03/093448; WO 04/009838; US2002/142370; US2003/040037; US2003/180835;US2004/063911; US2003/207406; and US2003/124645.

The invention provides methods for preparing conjugates of glycosylatedand unglycosylated mutant human growth hormones, which have newglycosylation sites that do not exist in the corresponding wild-typehGH. Such conjugation may take place directly on the appropriate sugarunits of a glycosylated mutant hGH, or following the removal (i.e.,“trimming back”) of any undesired sugar units. The conjugates are formedbetween peptides and diverse species such as water-soluble polymers,therapeutic moieties, diagnostic moieties, targeting moieties and thelike. Also provided are conjugates that include two or more peptideslinked together through a linker arm, i.e., multifunctional conjugates.The multi-functional conjugates of the invention can include two or morecopies of the same peptide or a collection of diverse peptides withdifferent structures, and/or properties.

The conjugates of the invention are formed by the enzymatic attachmentof a modified sugar to the glycosylated or unglycosylated peptide. Themodified sugar, when interposed between the peptide and the modifyinggroup on the sugar becomes what is referred to herein as “a glycosyllinking group,” e.g., an intact glycosyl linking group. Using theexquisite selectivity of enzymes, such as glycosyltransferases, thepresent method provides peptides that bear a desired group at one ormore specific locations. Thus, according to the present invention, amodified sugar is attached directly to a selected locus on the peptidechain or, alternatively, the modified sugar is appended onto acarbohydrate moiety of a glycopeptide. Peptides in which modified sugarsare bound to both a glycopeptide carbohydrate and directly to an aminoacid residue of the peptide backbone are also within the scope of thepresent invention.

In contrast to known chemical and enzymatic peptide elaborationstrategies, the methods of the invention make it possible to assemblepeptides and glycopeptides that have a substantially homogeneousderivatization pattern; the enzymes used in the invention are generallyselective for a particular amino acid residue or combination of aminoacid residues of the peptide. The methods are also practical forlarge-scale production of modified peptides and glycopeptides. Thus, themethods of the invention provide a practical means for large-scalepreparation of glycopeptides having preselected uniform derivatizationpatterns. The methods are particularly well suited for modification oftherapeutic peptides, including but not limited to, glycopeptides thatare incompletely glycosylated during production in cell culture cells(e.g., mammalian cells, insect cells, plant cells, fungal cells, yeastcells, or prokaryotic cells) or transgenic plants or animals.

The present invention also provides conjugates of glycosylated andunglycosylated peptides with increased therapeutic half-life due to, forexample, resistance to proteolysis, reduced clearance rate, or reducedrate of uptake by the immune or reticuloendothelial system (RES), etc.Moreover, the methods of the invention provide a means for maskingantigenic determinants on peptides, thus reducing or eliminating a hostimmune response against the peptide. Selective attachment of targetingagents can also be used to target a peptide to a particular tissue orcell surface receptor that is specific for the particular targetingagent.

The Conjugates

In a first aspect, the present invention provides a conjugate betweenone or more selected modifying groups, e.g. PEG, and a peptide that hasan in vivo activity similar or otherwise analogous to art-recognizedhGH. The modifying group(s) may be attached at a mutant glycosylationsite or at a site that is present in the wild type peptide.

As discussed herein, the selected modifying moiety is essentially anyspecies that can be attached to a saccharide unit, resulting in amodified sugar that is recognized by an appropriate transferase enzyme,which appends the modified sugar onto the peptide, or a glycosyl residueattached thereto. The saccharide component of the modified sugar, wheninterposed between the peptide and a selected moiety, becomes a“glycosyl linking group,” e.g., an “intact glycosyl linking group.” Theglycosyl linking group is formed from any mono-or oligo-saccharide that,after modification with the modifying group, is a substrate for anenzyme that adds the modified sugar to an amino acid or glycosyl residueof a peptide.

In some exemplary embodiments of the invention, the hGH peptide isconjugated to one or more polymeric modifying moieties through aglycosyl linking group. The polymeric modifying moiety can be attachedat any position of a glycosyl moiety of hGH. Moreover, the polymericmodifying moiety can be bound to a glycosyl residue at any position of awild type or mutant hGH amino acid sequence. Those of ordinary skill inthe art will therefore appreciate that an hGH peptide can be conjugatedto a plurality of the same or different polymeric modifying moieties viaa single glycosyl linking group or via multiple glycosyl linking groups.

Exemplary hGH peptide conjugates include a glycosyl linking group havinga formula selected from:

In Formulae A and B, R² is H, CH₂OR⁷, COOR⁷, COO⁻M⁺ or OR⁷, in which R⁷represents H, substituted or unsubstituted alkyl or substituted orunsubstituted heteroalkyl. When COOR⁷ is a carboxylic acid orcarboxylate, both forms are represented by the designation of the singlestructure COO⁻or COOH. The symbols R³, R⁴, R⁵, R⁶ and R^(6′)independently represent H, substituted or unsubstituted alkyl, OR⁸,NHC(O)R⁹. M⁺is a metal. The index d is 0 or 1. R⁸ and R⁹ areindependently selected from H, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, sialic acid or polysialicacid. At least one of R³, R⁴, R⁵, R⁶ or R^(6′) includes a polymericmodifying moiety e.g., PEG. In an exemplary embodiment, R⁶ and R^(6′),together with the carbon to which they are attached are components ofthe pyruvyl side chain of sialic acid. In a further exemplaryembodiment, this side chain is functionalized with the polymericmodifying moiety. In another exemplary embodiment, R⁶ and R^(6′),together with the carbon to which they are attached are components ofthe side chain of sialic acid and the polymeric modifying moiety is acomponent of R⁵.

In exemplary embodiments of the present invention, the polymericmodifying moiety is bound to the glycosyl linking group, generallythrough a heteroatom on the glycosyl core (e.g., N, O), through alinker, L, as shown below:

R¹ is the polymeric modifying group and L is selected from a bond and alinking group. The index w represents an integer selected from 1-6,preferably 1-3 and more preferably 1-2. Exemplary linking groups includesubstituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl moieties and sialic acid. An exemplary component of thelinker is an acyl moiety. Another exemplary linking group is an aminoacid residue (e.g., cysteine, serine, lysine, and short oligopeptides,e.g., Lys-Lys, Lys-Lys-Lys, Cys-Lys, Ser-Lys, etc.)

When L is a bond, it is formed by reaction of a reactive functionalgroup on a precursor of R¹ and a reactive functional group ofcomplementary reactivity on a precursor of the glycosyl linking group.When L is a non-zero order linking group, L can be in place on theglycosyl moiety prior to reaction with the R¹ precursor. Alternatively,the precursors of R¹ and L can be incorporated into a preformed cassettethat is subsequently attached to the glycosyl moiety.

An exemplary compound according to the invention has a structureaccording to Formulae A or B, in which at least one of R², R³, R⁴, R⁵,R⁶ or R^(6′) has the formula:

In another example according to this embodiment at least one of R², R³,R⁴, R⁵, R⁶ or R^(6′) has the formula:

in which s is an integer from 0 to 20 and R¹ is a linear polymericmodifying moiety.

In an exemplary embodiment, the polymeric modifying moiety-linkerconstruct is a branched structure that includes two or more polymericchains attached to central moiety. In this embodiment, the construct hasa structure according to Formula I.

When L is a bond it is formed between a reactive functional group on aprecursor of R¹ and a reactive functional group of complementaryreactivity on the saccharyl core. When L is a non-zero order linker, aprecursor of L can be in place on the glycosyl moiety prior to reactionwith the R¹ precursor. Alternatively, the precursors of R¹ and L can beincorporated into a preformed cassette that is subsequently attached tothe glycosyl moiety. As set forth herein, the selection and preparationof precursors with appropriate reactive functional groups is within theability of those skilled in the art. Moreover, coupling the precursorsproceeds by chemistry that is well understood in the art.

In an exemplary embodiment, L is a linking group that is formed from anamino acid, or small peptide (e.g., 1-4 amino acid residues) providing amodified sugar in which the polymeric modifying moiety is attachedthrough a substituted alkyl linker. Exemplary linkers include glycine,lysine, serine and cysteine. The PEG moiety can be attached to the aminemoiety of the linker through an amide or urethane bond. The PEG islinked to the sulfur or oxygen atoms of cysteine and serine throughthioether or ether bonds, respectively.

In an exemplary embodiment, R⁵ includes the polymeric modifying moiety.In another exemplary embodiment, R⁵ includes both the polymericmodifying moiety and a linker, L, joining the modifying moiety to theremainder of the molecule. As discussed above, L can be a linear orbranched structure. Similarly, the polymeric modifying moiety can bebranched or linear.

In an exemplary embodiment, Formula I has a structure according to thefollowing formula:

in which the moiety:

is the linker arm, L, and R¹⁶ and R¹⁷ are R¹. R¹⁶ and R¹⁷ areindependently selected polymeric modifying moieties. C is carbon. X⁵ ispreferably a non-reactive group (e.g., H, unsubstituted alkyl,unsubstituted heteroalkyl), and can be a polymeric arm. X² and X⁴ arelinkage fragments that are preferably essentially non-reactive underphysiological conditions, which may be the same or different. Anexemplary linker includes neither aromatic nor ester moieties.Alternatively, these linkages can include one or more moiety that isdesigned to degrade under physiologically relevant conditions, e.g.,esters, disulfides, etc. X² and X⁴ join polymeric arms R¹⁶ and R¹⁷ to C.Exemplary linkage fragments for X², X³ and X⁴ are independently selectedand include S, SC(O)NH, HNC(O)S, SC(O)O, O, NH, NHC(O), (O)CNH andNHC(O)O, and OC(O)NH, CH₂S, CH₂O, CH₂CH₂O, CH₂CH₂S, (CH₂)_(o)O,(CH₂)_(o)S or (CH₂)_(o)Y′-PEG wherein, Y′ is S, NH, NHC(O), C(O)NH,NHC(O)O, OC(O)NH, or O and o is an integer from 1 to 50. In an exemplaryembodiment, the linkage fragments X² and X⁴ are different linkagefragments.

In an exemplary embodiment, Formula I has a structure according to thefollowing formula:

the indices m and n are integers independently selected from 0 to 5000.A¹, A², A³, A⁴, A⁵, A⁶, A⁷, A⁸, A⁹, A¹⁰ and A¹¹ are membersindependently selected from H, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, substituted or unsubstituted heteroaryl,—NA¹²A¹³, —OA¹² and —SiA¹²A¹³. The indices j and k are integersindependently selected from 0 to 20. A¹² and A¹³ are membersindependently selected from substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, and substituted or unsubstituted heteroaryl.

In an exemplary embodiment, Formula IV has a structure according to thefollowing formula:

In an exemplary embodiment, A¹ and A² are each —OCH₃ or H.

In one embodiment, the present invention provides an hGH peptidecomprising the moiety:

wherein D is a member selected from —OH and R¹-L-HN—; G is a memberselected from H and R¹-L- and —C(O)(C₁-C₆)alkyl; R¹ is a moietycomprising a straight-chain or branched poly(ethylene glycol) residue;and L is a linker, e.g., a bond (“zero order”), substituted orunsubstituted alkyl and substituted or unsubstituted heteroalkyl. Inexemplary embodiments, when D is OH, G is R¹-L-, and when G is—C(O)(C₁-C₆)alkyl, D is R¹-L-NH—.

As set forth herein, the selection and preparation of precursors withappropriate reactive functional groups is within the ability of thoseskilled in the art. Moreover, coupling of the precursors proceeds bychemistry that is well understood in the art.

In an exemplary embodiment, the selected modifying moiety is awater-soluble polymer, e.g., m-PEG. The PEG moiety is attached directlyto a glycosyl linker, preferably an intact glycosyl linker, or via anon-glycosyl linker, e.g., substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl. The glycosyl linker iscovalently attached to an amino acid residue or a glycosyl residue ofthe peptide. The invention also provides conjugates in which an aminoacid residue and a glycosyl residue are modified with a glycosyl linker.

An exemplary water-soluble polymer is poly(ethylene glycol), e.g.,methoxy-poly(ethylene glycol). The poly(ethylene glycol) used in thepresent invention is not restricted to any particular form or molecularweight range. As discussed herein, the PEG of use in the conjugates ofthe invention can be linear or branched. For unbranched poly(ethyleneglycol) molecules, the molecular weight preferably ranges from about 500to 100,000. A molecular weight of 2000-60,000 is preferably used andpreferably of from 5,000 to about 30,000. More preferably, the molecularweight is from about 5,000 to about 30,000.

In another embodiment the poly(ethylene glycol) is a branched PEG havingmore than one PEG moiety attached. Examples of branched PEGs aredescribed in U.S. Pat. No. 5,932,462; U.S. Pat. No. 5,342,940; U.S. Pat.No. 5,643,575; U.S. Pat. No. 5,919,455; U.S. Pat. No. 6,113,906; U.S.Pat. No. 5,183,660; WO 02/09766; Kodera Y., Bioconjugate Chemistry 5:283-288 (1994); and Yamasaki et al., Agric. Biol. Chem., 52: 2125-2127,1998. In preferred embodiments of the present invention, the molecularweight of each poly(ethylene glycol) of the branched PEG is less than orequal to 40,000 daltons.

An exemplary precursor of use to form the branched PEG containingpeptide conjugates according to this embodiment of the invention has theformula:

The branched polymer species according to this formula are essentiallypure water-soluble polymers. X^(3′) is a moiety that includes anionizable (e.g., OH, COOH, H₂PO₄, HSO₃, NH₂, and salts thereof, etc.) orother reactive functional group, e.g., infra. C is carbon. X⁵, R¹⁶ andR¹⁷ are independently selected from non-reactive groups (e.g., H,unsubstituted alkyl, unsubstituted heteroalkyl) and polymeric arms(e.g., PEG). X² and X⁴ are linkage fragments that are preferablyessentially non-reactive under physiological conditions, which may bethe same or different. An exemplary linker includes neither aromatic norester moieties. Alternatively, these linkages can include one or moremoieties that are designed to degrade under physiologically relevantconditions, e.g., esters, disulfides, etc. X² and X⁴ join polymeric armsR¹⁶ and R¹⁷ to C. When X^(3′) is reacted with a reactive functionalgroup of complementary reactivity on a linker, sugar or linker-sugarcassette, X^(3′) is converted to a component of linkage fragment X³.

With respect to the glycosyl linking group, it can be, or can include, asaccharide moiety that is degradatively modified before or during theaddition of the modifying group. For example, the glycosyl linking groupcan be derived from a saccharide residue that is produced by oxidativedegradation of an intact saccharide to the corresponding aldehyde, e.g.,via the action of metaperiodate, and subsequently converted to a Schiffbase with an appropriate amine, which is then reduced to thecorresponding amine. Exemplary intact glycosyl linking groups includesialic acid moieties that are derivatized with PEG.

Exemplary conjugates of the present invention can be characterized bythe general structure:

in which the symbols a, b, c, and d represent a positive, non-zerointeger and s and t are either 0 or a positive integer. The “agent” is atherapeutic agent, a bioactive agent, a detectable label, water-solublemoiety (e.g., PEG, m-PEG, PPG, and m-PPG) or the like. The “agent” canbe a peptide, e.g., enzyme, antibody, antigen, etc. The linker can beany of a wide array of linking groups, infra. Alternatively, the linkermay be a single bond or a “zero order linker.”

In addition to providing conjugates that are formed through anenzymatically added glycosyl linking group, the present inventionprovides conjugates that are highly homogenous in their substitutionpatterns. Using the methods of the invention, it is possible to formpeptide conjugates in which essentially all of the modified sugarmoieties across a population of conjugates of the invention are attachedto multiple copies of a structurally identical amino acid or glycosylresidue. Thus, in a second aspect, the invention provides a peptideconjugate having a population of water-soluble polymer moieties, whichare covalently bound to the peptide through a glycosyl linking group,e.g. an intact glycosyl linking group. In a preferred conjugate of theinvention, essentially each member of the population is bound via theglycosyl linking group to a glycosyl residue of the peptide, and eachglycosyl residue of the peptide to which the glycosyl linking group isattached has the same structure.

Also provided is a peptide conjugate having a population ofwater-soluble polymer moieties covalently bound thereto through aglycosyl linking group. In a preferred embodiment, essentially everymember of the population of water soluble polymer moieties is bound toan amino acid residue of the peptide via a glycosyl linking group, andeach amino acid residue having a glycosyl linking group attached theretohas the same structure.

The present invention also provides conjugates analogous to thosedescribed above in which the peptide is conjugated to a therapeuticmoiety, diagnostic moiety, targeting moiety, toxin moiety or the likevia an intact glycosyl linking group. Each of the above-recited moietiescan be a small molecule, natural polymer (e.g., polypeptide) orsynthetic polymer.

The peptides of the invention include at least one N-, or O-linkedglycosylation site, which is glycosylated with a glycosyl residue thatincludes a polymeric modifying moiety, e.g. a PEG moiety. In exemplaryembodiments, the PEG is covalently attached to the peptide via an intactglycosyl linking group. The glycosyl linking group is covalentlyattached to either an amino acid residue or a glycosyl residue of thepeptide. Alternatively, the glycosyl linking group is attached to one ormore glycosyl units of a glycopeptide. The invention also providesconjugates in which a glycosyl linking group is attached to both anamino acid residue and a glycosyl residue.

In an exemplary embodiment, mutant human growth hormone is conjugated totransferrin via a bifunctional linker that includes an intact glycosyllinking group at each terminus of the PEG moiety. For example, oneterminus of the PEG linker is functionalized with an intact sialic acidlinker that is attached to transferrin and the other is functionalizedwith an intact GalNAc linker that is attached to the mutant hGH.

The conjugates of the invention can include intact glycosyl linkinggroups that are mono- or multi-valent (e.g., antennary structures).Thus, conjugates of the invention include both species in which aselected moiety is attached to a peptide via a monovalent glycosyllinking group. Also included within the invention are conjugates inwhich more than one selected moiety is attached to a peptide via amultivalent linking group.

In a still further embodiment, the invention provides conjugates thatlocalize selectively in a particular tissue due to the presence of atargeting agent as a component of the conjugate. In an exemplaryembodiment, the targeting agent is a protein. Exemplary proteins includetransferrin (brain, blood pool), HS-glycoprotein (bone, brain, bloodpool), antibodies (brain, tissue with antibody-specific antigen, bloodpool), coagulation factors V-XII (damaged tissue, clots, cancer, bloodpool), serum proteins, e.g., α-acid glycoprotein, fetuin, α-fetalprotein (brain, blood pool), β2-glycoprotein (liver, atherosclerosisplaques, brain, blood pool), hGH, GM-CSF, M-CSF, and EPO (immunestimulation, cancers, blood pool, red blood cell overproduction,neuroprotection), albumin (increase in half-life), and lipoprotein E.

The Methods

In addition to the conjugates discussed above, the present inventionprovides methods for preparing these and other conjugates. Thus, in afurther aspect, the invention provides a method of forming a covalentconjugate between a selected moiety and a peptide. Additionally, theinvention provides methods for targeting conjugates of the invention toa particular tissue or region of the body. Furthermore, the presentinvention provides a method for preventing, curing, or ameliorating adisease state by administering a conjugate of the invention to a subjectat risk of developing the disease or a subject that has the disease.

In exemplary embodiments, the conjugate is formed between a polymericmodifying moiety, a therapeutic moiety, targeting moiety or abiomolecule, and a glycosylated or non-glycosylated peptide. Thepolymer, therapeutic moiety or biomolecule is conjugated to the peptidevia a glycosyl linking group (or glycosyl residue), which is interposedbetween, and covalently linked to both the peptide and the modifyinggroup (e.g., water-soluble polymer). The method includes contacting thepeptide with a mixture containing a modified sugar and an enzyme, e.g.glycosyltransferase, that conjugates the modified sugar to thesubstrate. The reaction is conducted under conditions sufficient to forma covalent bond between the modified sugar and the peptide. The sugarmoiety of the modified sugar is preferably selected from nucleotidesugars, activated sugars, and sugars that are neither nucleotides noractivated.

The acceptor hGH peptide (glycosylated or non-glycosylated) is typicallysynthesized de novo, or recombinantly expressed in a prokaryotic cell(e.g., bacterial cell, such as E. coli) or in a eukaryotic cell such asa mammalian, yeast, insect, fungal or plant cell. The peptide can beeither a full-length protein or a fragment. Moreover, the peptide can bea wild type or mutated peptide. In an exemplary embodiment, the peptideincludes a mutation that adds one or more O- or N-linked glycosylationsites to the hGH peptide sequence.

The method of the invention also provides for modification ofincompletely glycosylated peptides that are produced recombinantly. Manyrecombinantly produced glycoproteins are incompletely glycosylated,exposing carbohydrate residues that may have undesirable properties,e.g., immunogenicity, recognition by the RES. Employing a modified sugarin a method of the invention, the peptide can be simultaneously furtherglycosylated and derivatized with, e.g., a water-soluble polymer,therapeutic agent, or the like. The sugar moiety of the modified sugarcan be the residue that would properly be conjugated to the acceptor ina fully glycosylated peptide, or another sugar moiety with desirableproperties.

Peptides modified by the methods of the invention can be synthetic orwild-type peptides or they can be mutated peptides, produced by methodsknown in the art, such as site-directed mutagenesis. Glycosylation ofpeptides is typically either N-linked or O-linked. An exemplaryN-linkage is the attachment of the modified sugar to the side chain ofan asparagine residue. The tripeptide sequences asparagine-X-serine andasparagine-X-threonine, where X is any amino acid except proline, arethe recognition sequences for enzymatic attachment of a carbohydratemoiety to the asparagine side chain. Thus, the presence of either ofthese tripeptide sequences in a polypeptide creates a potentialglycosylation site. O-linked glycosylation refers to the attachment ofone sugar (e.g., N-aceylgalactosamine, galactose, mannose, GlcNAc,glucose, fucose or xylose) to the hydroxy side chain of a hydroxyaminoacid, preferably serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to a peptide or other structure isconveniently accomplished by altering the amino acid sequence such thatit contains one or more glycosylation sites. The addition may also bemade by the incorporation of one or more species presenting an —OHgroup, preferably serine or threonine residues, within the sequence ofthe peptide (for O-linked glycosylation sites). The addition may be madeby mutation or by full chemical synthesis of the peptide. The peptideamino acid sequence is preferably altered through changes at the DNAlevel, particularly by mutating the DNA encoding the peptide atpreselected bases such that codons are generated that will translateinto the desired amino acids. The DNA mutation(s) are preferably madeusing methods known in the art.

In an exemplary embodiment, the glycosylation site is added by shufflingpolynucleotides. Polynucleotides encoding a candidate peptide can bemodulated with DNA shuffling protocols. DNA shuffling is a process ofrecursive recombination and mutation, performed by random fragmentationof a pool of related genes, followed by reassembly of the fragments by apolymerase chain reaction-like process. See, e.g., Stemmer, Proc. Natl.Acad. Sci. USA 91:10747-10751 (1994); Stemmer, Nature 370:389-391(1994); and U.S. Pat. Nos. 5,605,793, 5,837,458, 5,830,721 and5,811,238.

The present invention also provides means of adding (or removing) one ormore selected glycosyl residues to a peptide, after which a modifiedsugar is conjugated to at least one of the selected glycosyl residues ofthe peptide. The present embodiment is useful, for example, when it isdesired to conjugate the modified sugar to a selected glycosyl residuethat is either not present on a peptide or is not present in a desiredamount. Thus, prior to coupling a modified sugar to a peptide, theselected glycosyl residue is conjugated to the peptide by enzymatic orchemical coupling. In another embodiment, the glycosylation pattern of aglycopeptide is altered prior to the conjugation of the modified sugarby the removal of a carbohydrate residue from the glycopeptide. See, forexample WO 98/31826.

Addition or removal of any carbohydrate moieties present on theglycopeptide is accomplished either chemically or enzymatically.Chemical deglycosylation is preferably brought about by exposure of thepolypeptide variant to the compound trifluoromethanesulfonic acid, or anequivalent compound. This treatment results in the cleavage of most orall sugars except the linking sugar (N-acetylglucosamine orN-acetylgalactosamine), while leaving the peptide intact. Chemicaldeglycosylation is described by Hakimuddin et al., Arch. Biochem.Biophys. 259: 52 (1987) and by Edge et al., Anal. Biochem. 118: 131(1981). Enzymatic cleavage of carbohydrate moieties on polypeptidevariants can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al., Meth. Enzymol. 138:350 (1987).

Chemical addition of glycosyl moieties is carried out by anyart-recognized method. Enzymatic addition of sugar moieties ispreferably achieved using a modification of the methods set forthherein, substituting native glycosyl units for the modified sugars usedin the invention. Other methods of adding sugar moieties are disclosedin U.S. Pat. Nos. 5,876,980, 6,030,815, 5,728,554, and 5,922,577.

Exemplary attachment points for selected glycosyl residue include, butare not limited to: (a) consensus sites for N-linked glycosylation andO-linked glycosylation; (b) terminal glycosyl moieties that areacceptors for a glycosyltransferase; (c) arginine, asparagine andhistidine; (d) free carboxyl groups; (e) free sulfhydryl groups such asthose of cysteine; (f) free hydroxyl groups such as those of serine,threonine, or hydroxyproline; (g) aromatic residues such as those ofphenylalanine, tyrosine, or tryptophan; or (h) the amide group ofglutamine. Exemplary methods of use in the present invention aredescribed in WO 87/05330 published Sep. 11, 1987, and in Aplin andWriston, CRC CRIT. REV. BIOCHEM., pp. 259-306 (1981).

In an exemplary embodiment, the modified sugar, such as those set forthabove, is activated as the corresponding nucleotide sugars. Exemplarysugar nucleotides that are used in the present invention in theirmodified form include nucleotide mono-, di- or triphosphates or analogsthereof. In a preferred embodiment, the modified sugar nucleotide isselected from a UDP-glycoside, CMP-glycoside, or a GDP-glycoside. Evenmore preferably, the sugar nucleotide portion of the modified sugarnucleotide is selected from UDP-galactose, UDP-galactosamine,UDP-glucose, UDP-glucosamine, GDP-mannose, GDP-fucose, CMP-sialic acid,or CMP-NeuAc. In an exemplary embodiment, the nucleotide phosphate isattached to C-1.

Modified Sugars

The present invention uses modified sugars, such as modified sugarnucleotides, as reactants in the production of hGH conjugates. In themodified sugars of the invention, the sugar moiety is preferably asaccharide, a deoxy-saccharide, an amino-saccharide, or an N-acylsaccharide. The term “saccharide” and its equivalents, “saccharyl,”“sugar,” and “glycosyl” refer to monomers, dimers, oligomers andpolymers. The sugar moiety is also functionalized with a modifyinggroup. The modifying group is conjugated to the sugar moiety, typically,through conjugation with an amine, sulfhydryl or hydroxyl, e.g., primaryhydroxyl, moiety on the sugar. In an exemplary embodiment, the modifyinggroup is attached through an amine moiety on the sugar, e.g., through anamide, a urethane or a urea that is formed through the reaction of theamine with a reactive derivative of the modifying group.

Any sugar can be utilized in the hGH conjugates of the invention.Exemplary sugars that are useful in the conjugates of the inventioninclude, but are not limited to, glucose, galactose, mannose, fucose,and sialic acid. Other useful sugars include amino sugars such asglucosamine, galactosamine, mannosamine, the 5-amine analogue of sialicacid and the like. The sugar can be a structure found in nature or itcan be modified to provide a site for conjugating the modifying group.For example, in one embodiment, the invention provides a sialic acidderivative in which the 9-hydroxy moiety is replaced with an amine. Theamine is readily derivatized with an activated analogue of a modifyinggroup.

In an exemplary embodiment, the invention utilizes a modified sugaramine that has the formula:

in which J is a glycosyl moiety (e.g. a nucleotide sugar), and L is abond or a linker and R¹ is the modifying group, e.g. a polymericmodifying moiety. Exemplary bonds are those that are formed between anNH₂ moiety on the glycosyl moiety and a group of complementaryreactivity on the modifying group. For example, when R¹ includes acarboxylic acid moiety, such moiety may be activated and coupled withthe NH₂ moiety on the glycosyl residue, thereby affording a bond havingthe structure NHC(O)R¹. J is preferably a glycosyl moiety that is“intact,” i.e. not having been degraded by exposure to conditions thatcleave the pyranose or furanose structure, e.g. oxidative conditions,sodium periodate.

Exemplary linkers include alkyl and heteroalkyl moieties. The linkersinclude linking groups, such as acyl-based linking groups, e.g.,—C(O)NH—, —OC(O)NH—, and the like. The linking groups are bonds formedbetween components of the species of the invention, e.g. between theglycosyl moiety and the linker (L), or between the linker and themodifying group (R¹). Other exemplary linking groups are ethers,thioethers and amines. For example, in one embodiment, the linker is anamino acid residue, such as a glycine residue. The carboxylic acidmoiety of the glycine is converted to the corresponding amide byreaction with an amine on the glycosyl residue, and the amine of theglycine is converted to the corresponding amide or urethane by reactionwith an activated carboxylic acid or carbonate on the rest of themodifying group.

Another exemplary linker is a PEG moiety, e.g. a PEG moiety that isfunctionalized with an amino acid residue. The PEG linker is conjugatedto the glycosyl group through the amino acid residue at one PEG terminusand bound to R¹ through the other PEG terminus. Alternatively, the aminoacid residue is bound to R¹ and the PEG terminus, which is not bound tothe amino acid, is bound to the glycosyl group.

An exemplary species of NH— L-R¹ has the formula: —NH{C(O)(CH₂)_(a)NH}_(s){C(O)(CH₂)_(b)(OCH₂CH₂)_(c)O(CH₂)_(d)NH}_(t)R¹, inwhich the indices s and t are independently 0 or 1. The indices a, b andd are independently integers from 0 to 20, and c is an integer from 1 to2500. Other similar linkers are based on species in which an —NH moietyis replaced by another group, for example, —S, —O or —CH₂. As those ofskill will appreciate, one or more of the bracketed moietiescorresponding to indices s and t can be replaced with a substituted orunsubstituted alkyl or heteroalkyl moiety.

More particularly, the invention utilizes compounds in which NH-L⁵-R¹is:

NHC(O)(CH₂)_(a)NHC(O)(CH₂)_(b)(OCH₂CH₂)_(c)O(CH₂)_(d)NHR¹,

NHC(O)(CH₂)_(b)(OCH₂CH₂)_(c)O(CH₂)_(d)NHR¹,NHC(O)O(CH₂)_(b)(OCH₂CH₂)_(c)O(CH₂)_(d)NHR¹,

NH(CH₂)_(a)NHC(O)(CH₂)_(b)(OCH₂CH₂)_(c)O(CH₂)_(d)NHR¹,NHC(O)(CH₂)_(a)NHR¹,

NH(CH₂)_(a)NHR¹, and NHR¹. In these formulae, the indices a, b and d areindependently selected from the integers from 0 to 20, preferably from 1to 5. The index c is an integer from 1 to 2500.

In an exemplary embodiment, c is selected such that the PEG moiety isapproximately 1 kDa, 2 kDa, 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, 60 kDa, 65 kDa, 70 kDa, 75kDa or 80 kDa.

In the discussion that follows the invention is illustrated by referenceto the use of selected derivatives of sialic acid. Those of skill in theart will recognize that the focus of the discussion is for clarity ofillustration and that the structures and compositions set forth aregenerally applicable across the genus of saccharide groups, modifiedsaccharide groups, activated modified saccharide groups and conjugatesof modified saccharide groups.

In an exemplary embodiment, J is sialic acid and selected compounds ofuse in the invention have the formulae:

As those of skill in the art will appreciate, the sialic acid moiety inthe exemplary compounds above can be replaced with any otheramino-saccharide including, but not limited to, glucosamine,galactosamine, mannosamine, their N-acetyl derivatives, and the like.

In another illustrative embodiment, a primary hydroxyl moiety of thesugar is functionalized with a modifying group. For example, the9-hydroxyl of sialic acid can be converted to the corresponding amineand functionalized to provide a modified sugar according to theinvention. Formulae according to this embodiment include:

Thus, in an illustrative embodiment in which the glycosyl moiety issialic acid, the method of the invention utilizes compounds having theformulae:

in which L-R¹ is as discussed above, and L¹-R¹ represents a linker boundto the modifying group. As with L, exemplary linker species according toL¹ include a bond, alkyl or heteroalkyl moieties.

Moreover, as discussed above, the present invention provides for the useof nucleotide sugars that are modified with a water-soluble polymer,which is either straight-chain or branched. For example, compoundshaving the formula shown below are of use to prepare conjugates withinthe scope of the present invention:

in which X⁴ is O or a bond.

Selected conjugates according to this motif are based on mannose,galactose or glucose, or on species having the stereochemistry ofmannose, galactose or glucose. The general formulae of these conjugatesare:

As discussed above, the invention provides saccharides bearing amodifying group, activated analogues of these species and conjugatesformed between species such as peptides and lipids and a modifiedsaccharide of the invention.

In another exemplary embodiment, the invention utilizes modified sugarsas set forth above that are activated as the corresponding nucleotidesugars. Exemplary sugar nucleotides that are used in the presentinvention in their modified form include nucleotide mono-, di- ortriphosphates or analogs thereof. In a preferred embodiment, themodified sugar nucleotide is selected from a UDP-glycoside,CMP-glycoside, or a GDP-glycoside. Even more preferably, the sugarnucleotide portion of the modified sugar nucleotide is selected fromUDP-galactose, UDP-galactosamine, UDP-glucose, UDP-glucosamine,GDP-mannose, GDP-fucose, CMP-sialic acid, or CMP-NeuAc. In an exemplaryembodiment, the nucleotide phosphate is attached to C-1.

The invention also provides for the use of sugar nucleotides modifiedwith L-R¹ at the 6-carbon position. Exemplary species according to thisembodiment include:

in which the R groups, and L, represent moieties as discussed above. Theindex “y” is 0, 1 or 2. In an exemplary embodiment, L is a bond betweenNH and R¹. The base is a nucleic acid base.

Exemplary nucleotide sugars of use in the invention in which the carbonat the 6-position is modified include species having the stereochemistryof GDP mannose, e.g.:

in which X⁵ is a bond or O. The index i represents 0 or 1. The index arepresents an integer from 1 to 20. The indices e and f independentlyrepresent integers from 1 to 2500. Q, as discussed above, is H orsubstituted or unsubstituted C₁-C₆ alkyl. As those of skill willappreciate, the serine derivative, in which S is replaced with O alsofalls within this general motif.

In a still further exemplary embodiment, the invention provides aconjugate in which the modified sugar is based on the stereochemistry ofUDP galactose. An exemplary nucleotide sugar of use in this inventionhas the structure:

In another exemplary embodiment, the nucleotide sugar is based on thestereochemistry of glucose. Exemplary species according to thisembodiment have the formulae:

In one embodiment, the invention provides a method for linking hGH andone or more peptide through a linking group. The linking group is of anyuseful structure and may be selected from straight-chain and branchedchain structures. Preferably, each terminus of the linker, which isattached to a peptide, includes a modified sugar (i.e., a nascent intactglycosyl linking group).

In an exemplary method of the invention, two peptides are linkedtogether via a linker moiety that includes a PEG linker. The constructconforms to the general structure set forth in the cartoon above. Asdescribed herein, the construct of the invention includes two intactglycosyl linking groups (i.e., s+t=1). The focus on a PEG linker thatincludes two glycosyl groups is for purposes of clarity and should notbe interpreted as limiting the identity of linker arms of use in thisembodiment of the invention.

Thus, a PEG moiety is functionalized at a first terminus with a firstglycosyl unit and at a second terminus with a second glycosyl unit. Thefirst and second glycosyl units are preferably substrates for differenttransferases, allowing orthogonal attachment of the first and secondpeptides to the first and second glycosyl units, respectively. Inpractice, the (glycosyl)¹-PEG-(glycosyl)² linker is contacted with thefirst peptide and a first transferase for which the first glycosyl unitis a substrate, thereby forming (peptide)¹-(glycosyl)¹-PEG-(glycosyl)².Glycosyltransferase and/or unreacted peptide is then optionally removedfrom the reaction mixture. The second peptide and a second transferasefor which the second glycosyl unit is a substrate are added to the(peptide)¹-(glycosyl)¹-PEG-(glycosyl)² conjugate, forming(peptide)¹-(glycosyl)¹-PEG-(glycosyl)²-(peptide)². Those of skill in theart will appreciate that the method outlined above is also applicable toforming conjugates between more than two peptides by, for example, theuse of a branched PEG, dendrimer, poly(amino acid), polysaccharide orthe like.

In an exemplary embodiment, human growth hormone is conjugated totransferrin via a bifunctional linker that includes an intact glycosyllinking group at each terminus of the PEG moiety (Scheme 1). The hGHconjugate has an in vivo half-life that is increased over that of hGHalone by virtue of the greater molecular sized of the conjugate.Moreover, the conjugation of hGH to transferrin serves to selectivelytarget the conjugate to the brain. For example, one terminus of the PEGlinker is functionalized with a CMP sialic acid and the other isfunctionalized with an UDP GalNAc. The linker is combined with hGH inthe presence of a GalNAc transferase, resulting in the attachment of theGalNAc of the linker arm to a serine and/or threonine residue on thehGH.

The processes described above can be carried through as many cycles asdesired, and is not limited to forming a conjugate between two peptideswith a single linker. Moreover, those of skill in the art willappreciate that the reactions functionalizing the intact glycosyllinking groups at the termini of the PEG (or other) linker with thepeptide can occur simultaneously in the same reaction vessel, or theycan be carried out in a step-wise fashion. When the reactions arecarried out in a step-wise manner, the conjugate produced at each stepis optionally purified from one or more reaction components (e.g.,enzymes, peptides).

A still further exemplary embodiment is set forth in Scheme 2. Scheme 2shows a method of preparing a conjugate that targets a selected protein,e.g., human growth hormone, to bone and increases the circulatoryhalf-life of the selected protein.

in which G is a glycosyl residue on an activated sugar moiety (e.g.,sugar nucleotide), which is converted to an intact glycosyl linker groupin the conjugate. When s is greater than 0, L is a saccharyl linkinggroup such as GalNAc, or GalNAc-Gal.

The use of reactive derivatives of PEG (or other linkers) to attach oneor more peptide moieties to the linker is within the scope of thepresent invention. The invention is not limited by the identity of thereactive PEG analogue. Many activated derivatives of poly(ethyleneglycol) are available commercially and in the literature. It is wellwithin the abilities of one of skill to choose, and synthesize ifnecessary, an appropriate activated PEG derivative with which to preparea substrate useful in the present invention. See, Abuchowski et al.Cancer Biochem. Biophys., 7: 175-186 (1984); Abuchowski et al., J. Biol.Chem., 252: 3582-3586 (1977); Jackson et al., Anal. Biochem., 165:114-127 (1987); Koide et al., Biochem Biophys. Res. Commun., 111:659-667 (1983)), tresylate (Nilsson et al., Methods Enzymol., 104: 56-69(1984); Delgado et al., Biotechnol. Appl. Biochem., 12: 119-128 (1990));N-hydroxysuccinimide derived active esters (Buckmann et al., Makromol.Chem., 182: 1379-1384 (1981); Joppich et al., Makromol. Chem., 180:1381-1384 (1979); Abuchowski et al., Cancer Biochem. Biophys., 7:175-186 (1984); Katre et al. Proc. Natl. Acad. Sci. U.S.A., 84:1487-1491 (1987); Kitamura et al., Cancer Res., 51: 4310-4315 (1991);Boccu et al., Z. Naturforsch., 38C: 94-99 (1983), carbonates (Zalipskyet al., POLY(ETHYLENE GLYCOL) CHEMISTRY: BIOTECHNICAL AND BIOMEDICALAPPLICATIONS, Harris, Ed., Plenum Press, New York, 1992, pp. 347-370;Zalipsky et al., Biotechnol. Appl. Biochem., 15: 100-114 (1992);Veronese et al., Appl. Biochem. Biotech., 11: 141-152 (1985)),imidazolyl formates (Beauchamp et al., Anal. Biochem., 131: 25-33(1983); Berger et al., Blood, 71: 1641-1647 (1988)), 4-dithiopyridines(Woghiren et al., Bioconjugate Chem., 4: 314-318 (1993)), isocyanates(Byun et al., ASAIO Journal, M649-M-653 (1992)) and epoxides (U.S. Pat.No. 4,806,595, issued to Noishiki et al., (1989). Other linking groupsinclude the urethane linkage between amino groups and activated PEG.See, Veronese, et al., Appl. Biochem. Biotechnol., 11: 141-152 (1985).

In another exemplary embodiment, the invention provides a method forextending the blood-circulation half-life of a selected peptide, inessence targeting the peptide to the blood pool, by conjugating thepeptide to a synthetic or natural polymer of a size sufficient to retardthe filtration of the protein by the glomerulus (e.g., albumin). See,Scheme 3. This embodiment of the invention is illustrated in Scheme 3 inwhich hGH is conjugated to albumin via a PEG linker using a combinationof chemical and enzymatic modification.

Thus, as shown in Scheme 3, a residue (e.g., amino acid side chain) ofalbumin is modified with a reactive PEG derivative, such asX-PEG-(CMP-sialic acid), in which X is an activating group (e.g., activeester, isothiocyanate, etc). The PEG derivative and hGH are combined andcontacted with a transferase for which CMP-sialic acid is a substrate.In a further illustrative embodiment, an 1-amine of lysine is reactedwith the N-hydroxysuccinimide ester of the PEG-linker to form thealbumin conjugate. The CMP-sialic acid of the linker is enzymaticallyconjugated to an appropriate residue on hGH, e.g., Gal or GalNAc,thereby forming the conjugate. Those of skill will appreciate that theabove-described method is not limited to the reaction partners setforth. Moreover, the method can be practiced to form conjugates thatinclude more than two protein moieties by, for example, utilizing abranched linker having more than two termini.

In other exemplary embodiments, the invention utilizes modified sugarsin which the 6-hydroxyl position is converted to the corresponding aminemoiety, which bears a modifying group such as those set forth above.Exemplary saccharyl groups that can be used as the core of thesemodified sugars include Gal, GalNAc, Glc, GlcNAc, Fuc, Xyl, Man, and thelike. A representative modified sugar according to this embodiment hasthe formula:

in which R³-R⁶ are members independently selected from H, OH, C(O)CH₃,NH, and NH C(O)CH₃. R⁷ is a link to another glycosyl residue(—O-glycosyl) or to an amino acid of the hGH peptide. R⁶ is OR¹, NHR¹ orNH-L-R¹, which is as described above.

Thus, in an illustrative embodiment in which the sugar moiety is sialicacid. In another exemplary embodiment, the modified sugar is based upona 6-amino-N-acetyl-glycosyl moiety. As shown below forN-acetylgalactosamine, the 6-amino-sugar moiety is readily prepared bystandard methods.

In the scheme above, the index n represents an integer from 1 to 2500,preferably from 10 to 1500, and more preferably from 10 to 1200. Thesymbol “A” represents an activating group, e.g., a halo, a component ofan activated ester (e.g., a N-hydroxysuccinimide ester), a component ofa carbonate (e.g., p-nitrophenyl carbonate) and the like. Preferredactivated leaving groups, for use in the present invention, are thosethat do not significantly sterically encumber the enzymatic transfer ofthe glyco side to the acceptor. Those of skill in the art willappreciate that other PEG-amide nucleotide sugars are readily preparedby this and analogous methods.

In other exemplary embodiments, the amide moiety is replaced by a groupsuch as a urethane or a urea.

Due to the versatility of the methods available for modifying glycosylresidues on a therapeutic peptide such as hGH, the glycosyl structureson the peptide conjugates of the invention have substantially anystructure. Moreover, the glycans can be O-linked or N-linked. Asexemplified in the discussion below, each of the pyranose and furanosederivatives discussed above can be a component of a glycosyl moiety of apeptide.

Some embodiments of the invention provide a modified hGH peptide thatincludes a glycosyl group having the formula:

In other embodiments, the group has the formula:

in which the index t is 0 or 1.

In a still further exemplary embodiment, the group has a structure whichis a member selected from the following formulae:

in which the index t is 0 or 1.

In yet another embodiment, the group has the formula:

in which the index p represents and integer from 1 to 10; and a iseither 0 or 1.

In still further embodiments, R¹ is a branched PEG, for example, one ofthose species set forth above. Illustrative compounds according to thisembodiment include:

in which X⁴ is a bond or O.

In other exemplary embodiments, the invention provides hGH peptideconjugates that includes a glycosyl linking group, such as those setforth above, that is covalently attached to an amino acid residue of thepeptide. In one embodiment according to this motif, the glycosyl linkingmoiety is linked to a galactose residue through a Sia residue:

An exemplary species according to this motif is prepared by conjugatingSia-L-R¹ to a terminal sialic acid of a glycan using an enzyme thatforms Sia-Sia bonds, e.g., CST-II, ST8Sia-II, ST8Sia-II1 and ST8Sia-IV.

In another exemplary embodiment, the sugar moiety is a sialic acidmoiety that has been oxidized and conjugated to a polymeric modifyingmoiety, such as is described in commonly assigned U.S. ProvisionalPatent Application No. 60/641,956 (Attorney Docket No. 040853-01-5150),filed Jan. 6, 2005.

In this embodiment, an exemplary linker is derived from a natural orunnatural amino acid, amino acid analogue or amino acid mimetic, or asmall peptide formed from one or more such species. For example, certainbranched polymers found in the compounds of the invention have theformula:

X^(a) is a linkage fragment that is formed by the reaction of a reactivefunctional group, e.g. X^(3′), on a precursor of the branched polymericmodifying moiety and a reactive functional group on the sugar moiety, ora precursor to a linker. For example, when X^(3′) is a carboxylic acid,it can be activated and bound directly to an amine group pendent from anamino-saccharide (e.g., Sia, GalNH₂, GlcNH₂, ManNH₂, etc.), forming anX^(a) that is an amide. Additional exemplary reactive functional groupsand activated precursors are described hereinbelow. The index crepresents an integer from 1 to 10. The other symbols have the sameidentity as those discussed above.

In another exemplary embodiment, X^(a) is a linking moiety formed withanother linker:

in which X^(b) is a second linkage fragment and is independentlyselected from those groups set forth for X^(a), and similar to L, L¹ isa bond, substituted or unsubstituted alkyl or substituted orunsubstituted heteroalkyl.

Exemplary species for X^(a) and X^(b) include S, SC(O)NH, HNC(O)S,SC(O)O, O, NH, NHC(O), C(O)NH and NHC(O)O, and OC(O)NH.

In another exemplary embodiment, X⁴ is a peptide bond to R¹⁷, which isan amino acid, di-peptide, e.g. Lys-Lys, or tri-peptide (e.g.Lys-Lys-Lys) in which the alpha-amine moiety(ies) and/or side chainheteroatom are modified with a polymeric modifying moiety.

In a further exemplary embodiment, the conjugates of the inventioninclude a moiety, e.g. an R¹⁵ moiety that has a formula that is selectedfrom:

in which the identity of the radicals represented by the various symbolsis the same as that discussed hereinabove. L^(a) is a bond or a linkeras discussed above for L and L¹, e.g. substituted or unsubstituted alkylor substituted or unsubstituted heteroalkyl moiety. In an exemplaryembodiment, L^(a) is a moiety of the side chain of sialic acid that isfunctionalized with the polymeric modifying moiety as shown. ExemplaryL^(a) moieties include substituted or unsubstituted alkyl chains thatinclude one or more OH or NH₂.

In yet another exemplary embodiment, the invention provides conjugateshaving a moiety, e.g. an R¹⁵ moiety with formula:

The identity of the radicals represented by the various symbols is thesame as that discussed hereinabove. As those of skill will appreciate,the linker arm in Formulae VII and VIII is equally applicable to othermodified sugars set forth herein. In exemplary embodiment, the speciesof Formulae VI and VII are the R¹⁵ moieties attached to the glycanstructures set forth herein.

In yet another exemplary embodiment, the hGH peptide includes an R¹⁵moiety with the formula:

in which the identities of the radicals are as discussed above. Anexemplary species for L^(a) is —(CH₂)_(j)C(O)NH(CH₂)_(h)C(O)NH—, inwhich the indices h and j are independently selected integers from 0 to10. A further exemplary species is —C(O)NH—. The indices j and k areintegers independently selected from 0 to 20. The indices m and n areintegers independently selected from 0 to 5000. A¹, A², A³, A⁴, A⁵, A⁶,A⁷, A⁸, A⁹, A¹⁰ and A¹¹ are members independently selected from H,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, —NA¹²A¹³, —OA¹² and —SiA¹²A¹³.A¹² and A¹³ are members independently selected from substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, and substituted orunsubstituted heteroaryl.

In a further exemplary embodiment, the modified sugar is anoligosaccharide having an antennary structure. In a preferredembodiment, one or more of the termini of the antennae bear themodifying moiety. When more than one modifying moiety is attached to anoligosaccharide having an antennary structure, the oligosaccharide isuseful to “amplify” the modifying moiety; each oligosaccharide unitconjugated to the peptide attaches multiple copies of the modifyinggroup to the peptide. The general structure of a typical chelate of theinvention as set forth in the drawing above, encompasses multivalentspecies resulting from preparing a conjugate of the invention utilizingan antennary structure. Many antennary saccharide structures are knownin the art, and the present method can be practiced with them withoutlimitation.

The embodiments of the invention set forth above are further exemplifiedby reference to species in which the polymer is a water-soluble polymer,particularly poly(ethylene glycol) (“PEG”), e.g., methoxy-poly(ethyleneglycol). Those of skill will appreciate that the focus in the sectionsthat follow is for clarity of illustration and the various motifs setforth using PEG as an exemplary polymer are equally applicable tospecies in which a polymer other than PEG is utilized.

PEG of any molecular weight, e.g., 1 kDa, 2 kDa, 5 kDa, 10 kDa, 15 kDa,20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, 60 kDa,65 kDa, 70 kDa, 75 kDa or 80 kDa can be used in the present invention.

In an exemplary embodiment, the R¹⁵ moiety has a formula that is amember selected from the group:

In each of the structures above, the linker fragment —NH(CH₂)_(a)— canbe present or absent.

In other exemplary embodiments, the conjugate includes an R¹⁵ moietyselected from the group:

In each of the formulae above, the indices e and f are independentlyselected from the integers from 1 to 2500. In further exemplaryembodiments, e and f are selected to provide a PEG moiety that is about1 kDa, 2 kDa, 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40kDa, 45 kDa, 50 kDa, 55 kDa, 60 kDa, 65 kDa, 70 kDa, 75 kDa or 80 kDa.The symbol Q represents substituted or unsubstituted alkyl (e.g., C₁-C₆alkyl, e.g., methyl), substituted or unsubstituted heteroalkyl or H.

Other branched polymers have structures based on di-lysine (Lys-Lys)peptides,

and tri-lysine peptides (Lys-Lys-Lys), e.g.:

In each of the figures above, e, f, f′, and f″ represent integersindependently selected from 1 to 2500. The indices q, q′ and q″represent integers independently selected from 1 to 20.

Exemplary modifying groups are discussed below. The modifying groups canbe selected for their ability to impart to a polypeptide one or moredesirable properties. Exemplary properties include, but are not limitedto, enhaced pharmacokinetics, enhanced pharmacodynamics, improvedbiodistribution, providing a polyvalent species, improved watersolubility, enhanced or diminished lipophilicity, and tissue targeting.

Exemplary modified sugars are modified with water-soluble orwater-insoluble polymers. Examples of useful polymer are furtherexemplified below.

Water-Soluble Polymers

Many water-soluble polymers are known to those of skill in the art andare useful in practicing the present invention. The term water-solublepolymer encompasses species such as saccharides (e.g., dextran, amylose,hyalouronic acid, poly(sialic acid), heparans, heparins, etc.);poly(amino acids), e.g., poly(aspartic acid) and poly(glutamic acid);nucleic acids; synthetic polymers (e.g., poly(acrylic acid),poly(ethers), e.g., poly(ethylene glycol); peptides, proteins, and thelike. The present invention may be practiced with any water-solublepolymer with the sole limitation that the polymer must include a pointat which the remainder of the conjugate can be attached.

Methods for activation of polymers can also be found in WO 94/17039,U.S. Pat. No. 5,324,844, WO 94/18247, WO 94/04193, U.S. Pat. No.5,219,564, U.S. Pat. No. 5,122,614, WO 90/13540, U.S. Pat. No.5,281,698, and more WO 93/15189, and for conjugation between activatedpolymers and peptides, e.g. Coagulation Factor VIII (WO 94/15625),hemoglobin (WO 94/09027), oxygen carrying molecule (U.S. Pat. No.4,412,989), ribonuclease and superoxide dismutase (Veronese at al., App.Biochem. Biotech. 11: 141-45 (1985)).

Preferred water-soluble polymers are those in which a substantialproportion of the polymer molecules in a sample of the polymer are ofapproximately the same molecular weight; such polymers are“homodisperse.”

The present invention is further illustrated by reference to apoly(ethylene glycol) conjugate. Several reviews and monographs on thefunctionalization and conjugation of PEG are available. See, forexample, Harris, Macronol. Chem. Phys. C25: 325-373 (1985); Scouten,Methods in Enzymology 135: 30-65 (1987); Wong et al., Enzyme Microb.Technol. 14: 866-874 (1992); Delgado et al., Critical Reviews inTherapeutic Drug Carrier Systems 9: 249-304 (1992); Zalipsky,Bioconjugate Chem. 6: 150-165 (1995); and Bhadra, et al., Pharmazie,57:5-29 (2002). Routes for preparing reactive PEG molecules and formingconjugates using the reactive molecules are known in the art. Forexample, U.S. Pat. No. 5,672,662 discloses a water soluble andisolatable conjugate of an active ester of a polymer acid selected fromlinear or branched poly(alkylene oxides), poly(oxyethylated polyols),poly(olefinic alcohols), and poly(acrylomorpholine).

U.S. Pat. No. 6,376,604 sets forth a method for preparing awater-soluble 1-benzotriazolylcarbonate ester of a water-soluble andnon-peptidic polymer by reacting a terminal hydroxyl of the polymer withdi(1-benzotriazoyl)carbonate in an organic solvent. The active ester isused to form conjugates with a biologically active agent such as aprotein or peptide.

WO 99/45964 describes a conjugate comprising a biologically active agentand an activated water soluble polymer comprising a polymer backbonehaving at least one terminus linked to the polymer backbone through astable linkage, wherein at least one terminus comprises a branchingmoiety having proximal reactive groups linked to the branching moiety,in which the biologically active agent is linked to at least one of theproximal reactive groups. Other branched poly(ethylene glycols) aredescribed in WO 96/21469, U.S. Pat. No. 5,932,462 describes a conjugateformed with a branched PEG molecule that includes a branched terminusthat includes reactive functional groups. The free reactive groups areavailable to react with a biologically active species, such as a proteinor peptide, forming conjugates between the poly(ethylene glycol) and thebiologically active species. U.S. Pat. No. 5,446,090 describes abifunctional PEG linker and its use in forming conjugates having apeptide at each of the PEG linker termini.

Conjugates that include degradable PEG linkages are described in WO99/34833; and WO 99/14259, as well as in U.S. Pat. No. 6,348,558. Suchdegradable linkages are applicable in the present invention.

The art-recognized methods of polymer activation set forth above are ofuse in the context of the present invention in the formation of thebranched polymers set forth herein and also for the conjugation of thesebranched polymers to other species, e.g., sugars, sugar nucleotides andthe like.

In other exemplary embodiments, the branched PEG is based upon acysteine, serine or di-lysine core. Thus, further exemplary branchedPEGs include:

The modified sugars are prepared by reacting the glycosyl core (or alinker on the core) with a polymeric modifying moiety (or a linker onthe polymeric modifying moiety). The discussion that follows providesexamples of selected polymeric modifying moieties of use in theinvention. For example, representative polymeric modifying moietiesinclude structures that are based on side chain-containing amino acids,e.g., serine, cysteine, lysine, and small peptides, e.g., lys-lys.

Those of skill will appreciate that the free amine in the di-lysinestructures can also be pegylated through an amide or urethane bond witha PEG moiety.

In yet another embodiment, the branched PEG moiety is based upon atri-lysine peptide. The tri-lysine can be mono-, di-, tri-, ortetra-PEGylated. Exemplary species according to this embodiment have theformulae:

in which e, f and f′ are independently selected integers from 1 to 2500;and q, q′ and q″ are independently selected integers from 1 to 20.

In exemplary embodiments of the invention, the PEG is m-PEG (5 kD, 10kD, 15 kD, 20 kD or 30 kD). An exemplary branched PEG species is aserine- or cysteine-(m-PEG)₂ in which the m-PEG is a 20 kD m-PEG. In anexemplary embodiment, the branched PEG is the cysteine residue shownabove with a 20 kD m-PEG attached to the sulfur and another attached tothe nitrogen.

As will be apparent to those of skill, the branched polymers of use inthe invention include variations on the themes set forth above. Forexample the di-lysine-PEG conjugate shown above can include threepolymeric subunits, the third bonded to the α-amine shown as unmodifiedin the structure above. Similarly, the use of a tri-lysinefunctionalized with three or four polymeric subunits labeled with thepolymeric modifying moiety in a desired manner is within the scope ofthe invention.

Exemplary PEG molecules that are activated with these and otheractivating group species and methods of making the activated PEGs areset forth in WO 04/083259.

Those of skill in the art will appreciate that one or more of the m-PEGarms of the branched polymers shown above can be replaced by a PEGmoiety with a different terminus, e.g., OH, COOH, NH₂, C₂-C₁₀-alkyl,etc. Moreover, the structures above are readily modified by insertingalkyl linkers (or removing carbon atoms) between the α-carbon atom andthe functional group of the amino acid side chain. Thus, “homo”derivatives and higher homologues, as well as lower homologues, arewithin the scope of cores for branched PEGs of use in the presentinvention.

The branched PEG species set forth herein are readily prepared bymethods such as that set forth in the scheme below:

in which X^(a) is O or S and r is an integer from 1 to 5. The indices eand f are independently selected integers from 1 to 2500. In anexemplary embodiment, one or both of these indices are selected suchthat the polymer is about 10 kD, 15 kD, or 20 kD in molecular weight.

Thus, according to this scheme, a natural or unnatural amino acid iscontacted with an activated m-PEG derivative, in this case the tosylate,forming 1 by alkylating the side-chain heteroatom X^(a). Themono-functionalized m-PEG amino acid is submitted to N-acylationconditions with a reactive m-PEG derivative, thereby assembling branchedm-PEG 2. As one of skill will appreciate, the tosylate leaving group canbe replaced with any suitable leaving group, e.g., halogen, mesylate,triflate, etc. Similarly, the reactive carbonate utilized to acylate theamine can be replaced with an active ester, e.g., N-hydroxysuccinimide,etc., or the acid can be activated in situ using a dehydrating agentsuch as dicyclohexylcarbodiimide, carbonyldiimidazole, etc.

In other exemplary embodiments, the urea moiety is replaced by a groupsuch as a amide.

Water-Insoluble Polymers

In another embodiment, analogous to those discussed above, the modifiedsugars comprise a water-insoluble polymer, rather than a water-solublepolymer. The conjugates of the invention may also include one or morewater-insoluble polymers. This embodiment of the invention isillustrated by the use of the conjugate as a vehicle with which todeliver a therapeutic peptide in a controlled manner. Polymeric drugdelivery systems are known in the art. See, for example, Dunn et al.,Eds. POLYMERIC DRUGS AND DRUG DELIVERY SYSTEMS, ACS Symposium SeriesVol. 469, American Chemical Society, Washington, D.C. 1991. Those ofskill in the art will appreciate that substantially any known drugdelivery system is applicable to the conjugates of the presentinvention.

The motifs forth above for R¹, L-R¹, R¹⁵, R^(15′) and other radicals areequally applicable to water-insoluble polymers, which may beincorporated into the linear and branched structures without limitationutilizing chemistry readily accessible to those of skill in the art.

Representative water-insoluble polymers include, but are not limited to,polyphosphazines, poly(vinyl alcohols), polyamides, polycarbonates,polyalkylenes, polyacrylamides, polyalkylene glycols, polyalkyleneoxides, polyalkylene terephthalates, polyvinyl ethers, polyvinyl esters,polyvinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes,polyurethanes, poly(methyl methacrylate), poly(ethyl methacrylate),poly(butyl methacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate),poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropylacrylate), poly(isobutyl acrylate), poly(octadecyl acrylate)polyethylene, polypropylene, poly(ethylene glycol), poly(ethyleneoxide), poly (ethylene terephthalate), poly(vinyl acetate), polyvinylchloride, polystyrene, polyvinyl pyrrolidone, pluronics andpolyvinylphenol and copolymers thereof.

Synthetically modified natural polymers of use in hGH conjugates of theinvention include, but are not limited to, alkyl celluloses,hydroxyalkyl celluloses, cellulose ethers, cellulose esters, andnitrocelluloses. Particularly preferred members of the broad classes ofsynthetically modified natural polymers include, but are not limited to,methyl cellulose, ethyl cellulose, hydroxypropyl cellulose,hydroxypropyl methyl cellulose, hydroxybutyl methyl cellulose, celluloseacetate, cellulose propionate, cellulose acetate butyrate, celluloseacetate phthalate, carboxymethyl cellulose, cellulose triacetate,cellulose sulfate sodium salt, and polymers of acrylic and methacrylicesters and alginic acid.

These and the other polymers discussed herein can be readily obtainedfrom commercial sources such as Sigma Chemical Co. (St. Louis, Mo.),Polysciences (Warrenton, PA.), Aldrich (Milwaukee, Wis.), Fluka(Ronkonkoma, NY), and BioRad (Richmond, Calif.), or else synthesizedfrom monomers obtained from these suppliers using standard techniques.

Representative biodegradable polymers of use in the hGH conjugates ofthe invention include, but are not limited to, polylactides,polyglycolides and copolymers thereof, poly(ethylene terephthalate),poly(butyric acid), poly(valeric acid), poly(lactide-co-caprolactone),poly(lactide-co-glycolide), polyanhydrides, polyorthoesters, blends andcopolymers thereof. Of particular use are compositions that form gels,such as those including collagen, pluronics and the like.

The polymers of use in the invention include “hybrid” polymers thatinclude water-insoluble materials having within at least a portion oftheir structure, a bioresorbable molecule. An example of such a polymeris one that includes a water-insoluble copolymer, which has abioresorbable region, a hydrophilic region and a plurality ofcrosslinkable functional groups per polymer chain.

For purposes of the present invention, “water-insoluble materials”includes materials that are substantially insoluble in water orwater-containing environments. Thus, although certain regions orsegments of the copolymer may be hydrophilic or even water-soluble, thepolymer molecule, as a whole, does not to any substantial measuredissolve in water.

For purposes of the present invention, the term “bioresorbable molecule”includes a region that is capable of being metabolized or broken downand resorbed and/or eliminated through normal excretory routes by thebody. Such metabolites or break down products are preferablysubstantially non-toxic to the body.

The bioresorbable region may be either hydrophobic or hydrophilic, solong as the copolymer composition as a whole is not renderedwater-soluble. Thus, the bioresorbable region is selected based on thepreference that the polymer, as a whole, remains water-insoluble.Accordingly, the relative properties, i.e., the kinds of functionalgroups contained by, and the relative proportions of the bioresorbableregion, and the hydrophilic region are selected to ensure that usefulbioresorbable compositions remain water-insoluble.

Exemplary resorbable polymers include, for example, syntheticallyproduced resorbable block copolymers of poly(α-hydroxy-carboxylicacid)/poly(oxyalkylene, (see, Cohn et al., U.S. Pat. No. 4,826,945).These copolymers are not crosslinked and are water-soluble so that thebody can excrete the degraded block copolymer compositions. See, Youneset al., J. Biomed. Mater. Res. 21: 1301-1316 (1987); and Cohn et al., J.Biomed. Mater. Res. 22: 993-1009 (1988).

Presently preferred bioresorbable polymers include one or morecomponents selected from poly(esters), poly(hydroxy acids),poly(lactones), poly(amides), poly(ester-amides), poly(amino acids),poly(anhydrides), poly(orthoesters), poly(carbonates),poly(phosphazines), poly(phosphoesters), poly(thioesters),polysaccharides and mixtures thereof. More preferably still, thebioresorbable polymer includes a poly(hydroxy) acid component. Of thepoly(hydroxy) acids, polylactic acid, polyglycolic acid, polycaproicacid, polybutyric acid, polyvaleric acid and copolymers and mixturesthereof are preferred.

In addition to forming fragments that are absorbed in vivo(“bioresorbed”), preferred polymeric coatings for use in the methods ofthe invention can also form an excretable and/or metabolizable fragment.

Higher order copolymers can also be used in the present invention. Forexample, Casey et al., U.S. Pat. No. 4,438,253, which issued on Mar. 20,1984, discloses tri-block copolymers produced from thetransesterification of poly(glycolic acid) and an hydroxyl-endedpoly(alkylene glycol). Such compositions are disclosed for use asresorbable monofilament sutures. The flexibility of such compositions iscontrolled by the incorporation of an aromatic orthocarbonate, such astetra-p-tolyl orthocarbonate into the copolymer structure.

Other polymers based on lactic and/or glycolic acids can also beutilized. For example, Spinu, U.S. Pat. No. 5,202,413, which issued onApr. 13, 1993, discloses biodegradable multi-block copolymers havingsequentially ordered blocks of polylactide and/or polyglycolide producedby ring-opening polymerization of lactide and/or glycolide onto eitheran oligomeric diol or a diamine residue followed by chain extension witha di-functional compound, such as, a diisocyanate, diacylchloride ordichlorosilane.

Bioresorbable regions of coatings useful in the present invention can bedesigned to be hydrolytically and/or enzymatically cleavable. Forpurposes of the present invention, “hydrolytically cleavable” refers tothe susceptibility of the copolymer, especially the bioresorbableregion, to hydrolysis in water or a water-containing environment.Similarly, “enzymatically cleavable” as used herein refers to thesusceptibility of the copolymer, especially the bioresorbable region, tocleavage by endogenous or exogenous enzymes.

When placed within the body, the hydrophilic region can be processedinto excretable and/or metabolizable fragments. Thus, the hydrophilicregion can include, for example, polyethers, polyalkylene oxides,polyols, poly(vinyl pyrrolidine), poly(vinyl alcohol), poly(alkyloxazolines), polysaccharides, carbohydrates, peptides, proteins andcopolymers and mixtures thereof. Furthermore, the hydrophilic region canalso be, for example, a poly(alkylene)oxide. Such poly(alkylene)oxidescan include, for example, poly(ethylene)oxide, poly(propylene)oxide andmixtures and copolymers thereof.

Polymers that are components of hydrogels are also useful in the presentinvention. Hydrogels are polymeric materials that are capable ofabsorbing relatively large quantities of water. Examples of hydrogelforming compounds include, but are not limited to, polyacrylic acids,sodium carboxymethylcellulose, polyvinyl alcohol, polyvinyl pyrrolidine,gelatin, carrageenan and other polysaccharides,hydroxyethylenemethacrylic acid (HEMA), as well as derivatives thereof,and the like. Hydrogels can be produced that are stable, biodegradableand bioresorbable. Moreover, hydrogel compositions can include subunitsthat exhibit one or more of these properties.

Bio-compatible hydrogel compositions whose integrity can be controlledthrough crosslinking are known and are presently preferred for use inthe methods of the invention. For example, Hubbell et al., U.S. Pat. No.5,410,016, which issued on Apr. 25, 1995 and U.S. Pat. No. 5,529,914,which issued on Jun. 25, 1996, disclose water-soluble systems, which arecrosslinked block copolymers having a water-soluble central blocksegment sandwiched between two hydrolytically labile extensions. Suchcopolymers are further end-capped with photopolymerizable acrylatefunctionalities. When crosslinked, these systems become hydrogels. Thewater soluble central block of such copolymers can include poly(ethyleneglycol); whereas, the hydrolytically labile extensions can be apoly(α-hydroxy acid), such as polyglycolic acid or polylactic acid. See,Sawhney et al., Macromolecules 26: 581-587 (1993).

In another preferred embodiment, the gel is a thermoreversible gel.Thermoreversible gels including components, such as pluronics, collagen,gelatin, hyalouronic acid, polysaccharides, polyurethane hydrogel,polyurethane-urea hydrogel and combinations thereof are presentlypreferred.

In yet another exemplary embodiment, the hGH conjugates of the inventioninclude a component of a liposome. Liposomes can be prepared accordingto methods known to those skilled in the art, for example, as describedin Eppstein et al., U.S. Pat. No. 4,522,811, which issued on Jun. 11,1985. For example, liposome formulations may be prepared by dissolvingappropriate lipid(s) (such as stearoyl phosphatidyl ethanolamine,stearoyl phosphatidyl choline, arachadoyl phosphatidyl choline, andcholesterol) in an inorganic solvent that is then evaporated, leavingbehind a thin film of dried lipid on the surface of the container. Anaqueous solution of the active compound or its pharmaceuticallyacceptable salt is then introduced into the container. The container isthen swirled by hand to free lipid material from the sides of thecontainer and to disperse lipid aggregates, thereby forming theliposomal suspension.

The above-recited microparticles and methods of preparing themicroparticles are offered by way of example and they are not intendedto define the scope of microparticles of use in the present invention.It will be apparent to those of skill in the art that an array ofmicroparticles, fabricated by different methods, are of use in thepresent invention.

The structural formats discussed above in the context of thewater-soluble polymers, both straight-chain and branched, are generallyapplicable with respect to the water-insoluble polymers as well. Thus,for example, the cysteine, serine, dilysine, and trilysine branchingcores can be functionalized with two water-insoluble polymer moieties.The methods used to produce these species are generally closelyanalogous to those used to produce the water-soluble polymers.

The degree of PEG substitution of the modified sugars and hGH conjugatescan be controlled by choice of stoichiometry, number of availableglycosylation sites, selection of an enzyme that is selective for aparticular site, and the like, see U.S. Pat. App. No. 60/690,728.

Biomolecules

In another preferred embodiment, the modified sugar bears a biomolecule.In still further preferred embodiments, the biomolecule is a functionalprotein, enzyme, antigen, antibody, peptide, nucleic acid (e.g., singlenucleotides or nucleosides, oligonucleotides, polynucleotides andsingle- and higher-stranded nucleic acids), lectin, receptor or acombination thereof.

Preferred biomolecules are essentially non-fluorescent, or emit such aminimal amount of fluorescence that they are inappropriate for use as afluorescent marker in an assay. Moreover, it is generally preferred touse biomolecules that are not sugars. An exception to this preference isthe use of an otherwise naturally occurring sugar that is modified bycovalent attachment of another entity (e.g., PEG, biomolecule,therapeutic moiety, diagnostic moiety, etc.). In an exemplaryembodiment, a sugar moiety, which is a biomolecule, is conjugated to alinker arm and the sugar-linker arm cassette is subsequently conjugatedto a peptide via a method of the invention.

Biomolecules useful in practicing the present invention can be derivedfrom any source. The biomolecules can be isolated from natural sourcesor they can be produced by synthetic methods. Peptides can be naturalpeptides or mutated peptides. Mutations can be effected by chemicalmutagenesis, site-directed mutagenesis or other means of inducingmutations known to those of skill in the art. Peptides useful inpracticing the instant invention include, for example, enzymes,antigens, antibodies and receptors. Antibodies can be either polyclonalor monoclonal; either intact or fragments. The peptides are optionallythe products of a program of directed evolution.

Both naturally derived and synthetic peptides and nucleic acids are ofuse in conjunction with the present invention; these molecules can beattached to a sugar residue component or a crosslinking agent by anyavailable reactive group. For example, peptides can be attached througha reactive amine, carboxyl, sulfhydryl, or hydroxyl group. The reactivegroup can reside at a peptide terminus or at a site internal to thepeptide chain. Nucleic acids can be attached through a reactive group ona base (e.g., exocyclic amine) or an available hydroxyl group on a sugarmoiety (e.g., 3′- or 5′-hydroxyl). The peptide and nucleic acid chainscan be further derivatized at one or more sites to allow for theattachment of appropriate reactive groups onto the chain. See, Chriseyet al. Nucleic Acids Res. 24: 3031-3039 (1996).

In a further preferred embodiment, the biomolecule is selected to directthe peptide modified by the methods of the invention to a specifictissue, thereby enhancing the delivery of the peptide to that tissuerelative to the amount of underivatized peptide that is delivered to thetissue. In a still further preferred embodiment, the amount ofderivatized peptide delivered to a specific tissue within a selectedtime period is enhanced by derivatization by at least about 20%, morepreferably, at least about 40%, and more preferably still, at leastabout 100%. Presently, preferred biomolecules for targeting applicationsinclude antibodies, hormones and ligands for cell-surface receptors.

In still a further exemplary embodiment, there is provided as conjugatewith biotin. Thus, for example, a selectively biotinylated peptide iselaborated by the attachment of an avidin or streptavidin moiety bearingone or more modifying groups.

Therapeutic Moieties

In another preferred embodiment, the modified sugar includes atherapeutic moiety. Those of skill in the art will appreciate that thereis overlap between the category of therapeutic moieties andbiomolecules; many biomolecules have therapeutic properties orpotential.

The therapeutic moieties can be agents already accepted for clinical useor they can be drugs whose use is experimental, or whose activity ormechanism of action is under investigation. The therapeutic moieties canhave a proven action in a given disease state or can be onlyhypothesized to show desirable action in a given disease state. In apreferred embodiment, the therapeutic moieties are compounds, which arebeing screened for their ability to interact with a tissue of choice.Therapeutic moieties, which are useful in practicing the instantinvention include drugs from a broad range of drug classes having avariety of pharmacological activities. Preferred therapeutic moietiesare essentially non-fluorescent, or emit such a minimal amount offluorescence that they are inappropriate for use as a fluorescent markerin an assay. Moreover, it is generally preferred to use therapeuticmoieties that are not sugars. An exception to this preference is the useof a sugar that is modified by covalent attachment of another entity,such as a PEG, biomolecule, therapeutic moiety, diagnostic moiety andthe like. In another exemplary embodiment, a therapeutic sugar moiety isconjugated to a linker arm and the sugar-linker arm cassette issubsequently conjugated to a peptide via a method of the invention.

Methods of conjugating therapeutic and diagnostic agents to variousother species are well known to those of skill in the art. See, forexample Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, San Diego,1996; and Dunn et al., Eds. POLYMERIC DRUGS AND DRUG DELIVERY SYSTEMS ,ACS Symposium Series Vol. 469, American Chemical Society, Washington,D.C. 1991.

In an exemplary embodiment, the therapeutic moiety is attached to themodified sugar via a linkage that is cleaved under selected conditions.Exemplary conditions include, but are not limited to, a selected pH(e.g., stomach, intestine, endocytotic vacuole), the presence of anactive enzyme (e.g., esterase, reductase, oxidase), light, heat and thelike. Many cleaveable groups are known in the art. See, for example,Jung et al., Biochem. Biophys. Acta, 761: 152-162 (1983); Joshi et al.,J. Biol. Chem., 265: 14518-14525 (1990); Zarling et al., J. Immunol.,124: 913-920 (1980); Bouizar et al., Eur. J. Biochem., 155: 141-147(1986); Park et al., J. Biol. Chem., 261: 205-210 (1986); Browning etal., J. Immunol., 143: 1859-1867 (1989).

Enzymes

Glycosyltransferases

Glycosyltransferases catalyze the addition of activated sugars (donorNDP-sugars), in a step-wise fashion, to a protein, glycopeptide, lipidor glycolipid or to the non-reducing end of a growing oligosaccharide.N-linked glycopeptides are synthesized via a transferase and alipid-linked oligosaccharide donor Dol-PP-NAG₂Glc₃Man₉ in an en blocktransfer followed by trimming of the core. In this case the nature ofthe “core” saccharide is somewhat different from subsequent attachments.A very large number of glycosyltransferases are known in the art.

The glycosyltransferase to be used in the present invention may be anyas long as it can utilize the modified sugar as a sugar donor. Examplesof such enzymes include Leloir pathway glycosyltransferase, such asgalactosyltransferase, N-acetylglucosaminyltransferase,N-acetylgalactosaminyltransferase, fucosyltransferase,sialyltransferase, mannosyltransferase, xylosyltransferase,glucurononyltransferase and the like.

For enzymatic saccharide syntheses that involve glycosyltransferasereactions, glycosyltransferase can be cloned, or isolated from anysource. Many cloned glycosyltransferases are known, as are theirpolynucleotide sequences. See, e.g., “The WWW Guide To ClonedGlycosyltransferases,” (http://www.vei.co.uk/TGN/gt_guide.htm).Glycosyltransferase amino acid sequences and nucleotide sequencesencoding glycosyltransferases from which the amino acid sequences can bededuced are also found in various publicly available databases,including GenBank, Swiss-Prot, EMBL, and others.

Glycosyltransferases that can be employed in the methods of theinvention include, but are not limited to, galactosyltransferases,fucosyltransferases, glucosyltransferases,N-acetylgalactosaminyltransferases, N-acetylglucosaminyltransferases,glucuronyltransferases, sialyltransferases, mannosyltransferases,glucuronic acid transferases, galacturonic acid transferases, andoligosaccharyltransferases. Suitable glycosyltransferases include thoseobtained from eukaryotes, as well as from prokaryotes.

DNA encoding the enzyme glycosyltransferases may be obtained by chemicalsynthesis, by screening reverse transcripts of mRNA from appropriatecells or cell line cultures, by screening genomic libraries fromappropriate cells, or by combinations of these procedures. Screening ofmRNA or genomic DNA may be carried out with oligonucleotide probesgenerated from the glycosyltransferases gene sequence. Probes may belabeled with a detectable group such as a fluorescent group, aradioactive atom or a chemiluminescent group in accordance with knownprocedures and used in conventional hybridization assays. In thealternative, glycosyltransferases gene sequences may be obtained by useof the polymerase chain reaction (PCR) procedure, with the PCRoligonucleotide primers being produced from the glycosyltransferasesgene sequence. See, U.S. Pat. No. 4,683,195 to Mullis et al. and U.S.Pat. No. 4,683,202 to Mullis.

The glycosyltransferases enzyme may be synthesized in host cellstransformed with vectors containing DNA encoding theglycosyltransferases enzyme. A vector is a replicable DNA construct.Vectors are used either to amplify DNA encoding the glycosyltransferasesenzyme and/or to express DNA which encodes the glycosyltransferasesenzyme. An expression vector is a replicable DNA construct in which aDNA sequence encoding the glycosyltransferases enzyme is operably linkedto suitable control sequences capable of effecting the expression of theglycosyltransferases enzyme in a suitable host. The need for suchcontrol sequences will vary depending upon the host selected and thetransformation method chosen. Generally, control sequences include atranscriptional promoter, an optional operator sequence to controltranscription, a sequence encoding suitable mRNA ribosomal bindingsites, and sequences which control the termination of transcription andtranslation. Amplification vectors do not require expression controldomains. All that is needed is the ability to replicate in a host,usually conferred by an origin of replication, and a selection gene tofacilitate recognition of transformants.

Fucosyltransferases

In some embodiments, a glycosyltransferase used in the method of theinvention is a fucosyltransferase. Fucosyltransferases are known tothose of skill in the art. Exemplary fucosyltransferases includeenzymes, which transfer L-fucose from GDP-fucose to a hydroxy positionof an acceptor sugar. Fucosyltransferases that transfer non-nucleotidesugars to an acceptor are also of use in the present invention.

In some embodiments, the acceptor sugar is, for example, the GlcNAc in aGalβ(1→3,4)GlcNAcβ-group in an oligosaccharide glycoside. Suitablefucosyltransferases for this reaction include theGalβ(1→3,4)GlcNAcβ1-α(1→3,4)fucosyltransferase (FTIII E.C. No.2.4.1.65), which was first characterized from human milk (see, Palcic,et al., Carbohydrate Res. 190: 1-11 (1989); Prieels, et al., J. Biol.Chem. 256: 10456-10463 (1981); and Nunez, et al., Can. J. Chem. 59:2086-2095 (1981)) and the Galβ(1→4)GlcNAcβ-αfucosyltransferases (FTIV,FTV, FTVI) which are found in human serum. FTVII (E.C. No. 2.4.1.65), asialyl α(2→3)Galβ((1→3)GlcNAcβ fucosyltransferase, has also beencharacterized. A recombinant form of the Galβ(1→3,4)GlcNAcβ-α(1→3,4)fucosyltransferase has also been characterized (see,Dumas, et al., Bioorg. Med. Letters 1: 425-428 (1991) andKukowska-Latallo, et al., Genes and Development 4: 1288-1303 (1990)).Other exemplary fucosyltransferases include, for example, α1,2fucosyltransferase (E.C. No. 2.4.1.69). Enzymatic fucosylation can becarried out by the methods described in Mollicone, et al., Eur. J.Biochem. 191: 169-176 (1990) or U.S. Pat. No. 5,374,655. Cells that areused to produce a fucosyltransferase will also include an enzymaticsystem for synthesizing GDP-fucose.

Galactosyltransferases

In another group of embodiments, the glycosyltransferase is agalactosyltransferase. Exemplary galactosyltransferases include α(1,3)galactosyltransferases (E.C. No. 2.4.1.151, see, e.g., Dabkowski et al.,Transplant Proc. 25:2921 (1993) and Yamamoto et al. Nature 345: 229-233(1990), bovine (GenBank j04989, Joziasse et al., J. Biol. Chem. 264:14290-14297 (1989)), murine (GenBank m26925; Larsen et al., Proc. Nat'l.Acad. Sci. USA 86: 8227-8231 (1989)), porcine (GenBank L36152; Strahanet al., Immunogenetics 41: 101-105 (1995)). Another suitable α1,3galactosyltransferase is that which is involved in synthesis of theblood group B antigen (EC 2.4.1.37, Yamamoto et al., J. Biol. Chem. 265:1146-1151 (1990) (human)).

Also suitable for use in the methods of the invention are β(1,4)galactosyltransferases, which include, for example, EC 2.4.1.90 (LacNAcsynthetase) and EC 2.4.1.22 (lactose synthetase) (bovine (D'Agostaro etal., Eur. J. Biochem. 183: 211-217 (1989)), human (Masri et al.,Biochem. Biophys. Res. Commun. 157: 657-663 (1988)), murine (Nakazawa etal., J. Biochem. 104: 165-168 (1988)), as well as E.C. 2.4.1.38 and theceramide galactosyltransferase (EC 2.4.1.45, Stahl et al., J. Neurosci.Res. 38: 234-242 (1994)). Other suitable galactosyltransferases include,for example, α1,2 galactosyltransferases (from e.g., Schizosaccharomycespombe, Chapell et al., Mol. Biol. Cell 5: 519-528 (1994)).

The production of proteins such as the enzyme GalNAc T_(I-XIV) fromcloned genes by genetic engineering is well known. See, eg., U.S. Pat.No. 4,761,371. One method involves collection of sufficient samples,then the amino acid sequence of the enzyme is determined by N-terminalsequencing. This information is then used to isolate a cDNA cloneencoding a full-length (membrane bound) transferase which uponexpression in the insect cell line Sf9 resulted in the synthesis of afully active enzyme. The acceptor specificity of the enzyme is thendetermined using a semiquantitative analysis of the amino acidssurrounding known glycosylation sites in 16 different proteins followedby in vitro glycosylation studies of synthetic peptides. This work hasdemonstrated that certain amino acid residues are overrepresented inglycosylated peptide segments and that residues in specific positionssurrounding glycosylated serine and threonine residues may have a moremarked influence on acceptor efficiency than other amino acid moieties.

Sialyltransferases

Sialyltransferases are another type of glycosyltransferase that isuseful in the recombinant cells and reaction mixtures of the invention.Cells that produce recombinant sialyltransferases will also produceCMP-sialic acid, which is a sialic acid donor for sialyltransferases.Examples of sialyltransferases that are suitable for use in the presentinvention include ST3Gal III (e.g., a rat or human ST3Gal III), ST3GalIV, ST3Gal I, ST6Gal I, ST3Gal V, ST6Gal II, ST6GalNAc I, ST6GalNAc II,and ST6GalNAc III (the sialyltransferase nomenclature used herein is asdescribed in Tsuji et al., Glycobiology 6: v-xiv (1996)). An exemplaryα(2,3)sialyltransferase referred to as α(2,3)sialyltransferase (EC2.4.99.6) transfers sialic acid to the non-reducing terminal Gal of aGalβ1→3Glc disaccharide or glycoside. See, Van den Eijnden et al., J.Biol. Chem. 256: 3159 (1981), Weinstein et al., J. Biol. Chem. 257:13845 (1982) and Wen et al., J. Biol. Chem. 267: 21011 (1992). Anotherexemplary α2,3-sialyltransferase (EC 2.4.99.4) transfers sialic acid tothe non-reducing terminal Gal of the disaccharide or glycoside. see,Rearick et al., J. Biol. Chem. 254: 4444 (1979) and Gillespie et al., J.Biol. Chem. 267: 21004 (1992). Further exemplary enzymes includeGal-β-1,4-GlcNAc α-2,6 sialyltransferase (See, Kurosawa et al. Eur. J.Biochem. 219: 375-381 (1994)).

Preferably, for glycosylation of carbohydrates of glycopeptides thesialyltransferase will be able to transfer sialic acid to the sequenceGalβ1,4GlcNAc-, the most common penultimate sequence underlying theterminal sialic acid on fully sialylated carbohydrate structures (see,Table 3). TABLE 3 Sialyltransferases which use the Galβ1,4GlcNAcsequence as an acceptor substrate Sialyltransferase Source Sequence(s)formed Ref. ST6Gal I Mammalian NeuAcα2,6Galβ1,4GlCNAc- 1 ST3Gal IIIMammalian NeuAcα2,3Galβ1,4GlCNAc- 1 NeuAcα2,3Galβ1,3GlCNAc- ST3Gal IVMammalian NeuAcα2,3Galβ1,4GlCNAc- 1 NeuAcα2,3Galβ1,3GlCNAc- ST6Gal IIMammalian NeuAcα2,6Galβ1,4GlCNA ST6Gal II photobacteriumNeuAcα2,6Galβ1,4GlCNAc- 2 ST3Gal V N. meningitidesNeuAcα2,3Galβ1,4GlCNAc- 3 N. gonorrhoeaeGoochee et al., Bio/Technology 9: 1347-1355 (1991)Yamamoto et al., J. Biochem. 120: 104-110 (1996)Gilbert et al., J. Biol. Chem. 271: 28271-28276 (1996)

An example of a sialyltransferase that is useful in the claimed methodsis ST3Gal III, which is also referred to as α(2,3)sialyltransferase (EC2.4.99.6). This enzyme catalyzes the transfer of sialic acid to the Galof a Galβ1,3GlcNAc or Galβ1,4GlcNAc glycoside (see, e.g., Wen et al., J.Biol. Chem. 267: 21011 (1992); Van den Eijnden et al., J. Biol. Chem.256: 3159 (1991)) and is responsible for sialylation ofasparagine-linked oligosaccharides in glycopeptides. The sialic acid islinked to a Gal with the formation of an α-linkage between the twosaccharides. Bonding (linkage) between the saccharides is between the2-position of NeuAc and the 3-position of Gal. This particular enzymecan be isolated from rat liver (Weinstein et al., J. Biol. Chem. 257:13845 (1982)); the human cDNA (Sasaki et al. (1993) J. Biol. Chem. 268:22782-22787; Kitagawa & Paulson (1994) J. Biol. Chem. 269: 1394-1401)and genomic (Kitagawa et al. (1996) J. Biol. Chem. 271: 931-938) DNAsequences are known, facilitating production of this enzyme byrecombinant expression. In a preferred embodiment, the claimedsialylation methods use a rat ST3Gal III.

Other exemplary sialyltransferases of use in the present inventioninclude those isolated from Campylobacter jejuni, including the α(2,3).See, e.g., WO99/49051.

Sialyltransferases other than those listed in Table 3, are also usefulin an economic and efficient large-scale process for sialylation ofcommercially important glycopeptides. As a simple test to find out theutility of these other enzymes, various amounts of each enzyme (1-100mU/mg protein) are reacted with asialo-α₁ AGP (at 1-10 mg/ml) to comparethe ability of the sialyltransferase of interest to sialylateglycopeptides relative to either bovine ST6Gal I, ST3Gal III or bothsialyltransferases. Alternatively, other glycopeptides or glycopeptides,or N-linked oligosaccharides enzymatically released from the peptidebackbone can be used in place of asialo-α₁ AGP for this evaluation.Sialyltransferases with the ability to sialylate N-linkedoligosaccharides of glycopeptides more efficiently than ST6Gal I areuseful in a practical large-scale process for peptide sialylation (asillustrated for ST3Gal III in this disclosure).

Other Glycosyltransferases

One of skill in the art will understand that other glycosyltransferasescan be substituted into similar transferase cycles as have beendescribed in detail for the sialyltransferase. In particular, theglycosyltransferase can also be, for instance, glucosyltransferases,e.g., Alg8 (Stagljov et al., Proc. Natl. Acad. Sci. USA 91: 5977 (1994))or AlgS (Heesen et al., Eur. J. Biochem. 224: 71 (1994)).

N-acetylgalactosaminyltransferases are also of use in practicing thepresent invention. Suitable N-acetylgalactosaminyltransferases include,but are not limited to, α(1,3) N-acetylgalactosaminyltransferase, β(1,4)N-acetylgalactosaminyltransferases (Nagata et al., J. Biol. Chem. 267:12082-12089 (1992) and Smith et al., J. Biol. Chem. 269: 15162 (1994))and polypeptide N-acetylgalactosaminyltransferase (Homa et al., J. Biol.Chem. 268: 12609 (1993)). Suitable N-acetylglucosaminyltransferasesinclude GnTI (2.4.1.101, Hull et al., BBRC 176: 608 (1991)), GnTII,GnTIII (Ihara et al., J. Biochem. 113: 692 (1993)), GnTIV, and GnTV(Shoreiban et al., J. Biol. Chem. 268: 15381 (1993)), O-linkedN-acetylglucosaminyltransferase (Bierhuizen et al., Proc. Natl. Acad.Sci. USA 89: 9326 (1992)), N-acetylglucosamine-1-phosphate transferase(Rajput et al., Biochem J. 285: 985 (1992), and hyaluronan synthase.

Mannosyltransferases are of use to transfer modified mannose moieties.Suitable mannosyltransferases include α(1,2) mannosyltransferase, α(1,3)mannosyltransferase, α(1,6) mannosyltransferase, β(1,4)mannosyltransferase, Dol-P-Man synthase, OCh1, and Pmt1 (see, Kornfeldet al., Annu. Rev. Biochem. 54: 631-664 (1985)).

Xylosyltransferases are also useful in the present invention. See, forexample, Rodgers, et al., Biochem. J., 288:817-822 (1992); and Elbain,et al., U.S. Pat. No. 6,168,937.

Other suitable glycosyltransferase cycles are described in Ichikawa etal., JACS 114: 9283 (1992), Wong et al., J. Org. Chem. 57: 4343 (1992),and Ichikawa et al. in CARBOHYDRATES AND CARBOHYDRATE POLYMERS. Yaltami,ed. (ATL Press, 1993).

Prokaryotic glycosyltransferases are also useful in practicing theinvention. Such glycosyltransferases include enzymes involved insynthesis of lipooligosaccharides (LOS), which are produced by many gramnegative bacteria. The LOS typically have terminal glycan sequences thatmimic glycoconjugates found on the surface of human epithelial cells orin host secretions (Preston et al., Critical Reviews in Microbiology23(3): 139-180 (1996)). Such enzymes include, but are not limited to,the proteins of the rfa operons of species such as E. coli andSalmonella typhimurium, which include a β1,6 galactosyltransferase and aβ1,3 galactosyltransferase (see, e.g., EMBL Accession Nos. M80599 andM86935 (E. coli); EMBL Accession No. S56361 (S. typhimurium)), aglucosyltransferase (Swiss-Prot Accession No. P25740 (E. coli), anβ1,2-glucosyltransferase (rfaJ)(Swiss-Prot Accession No. P27129 (E.coli) and Swiss-Prot Accession No. P19817 (S. typhimurium)), and anβ1,2-N-acetylglucosaminyltransferase (rfaK)(EMBL Accession No. U00039(E. coli). Other glycosyltransferases for which amino acid sequences areknown include those that are encoded by operons such as rfaB, which havebeen characterized in organisms such as Klebsiella pneumoniae, E. coli,Salmonella typhimurium, Salmonella enterica, Yersinia enterocolitica,Mycobacterium leprosum, and the rh1 operon of Pseudomonas aeruginosa.

Also suitable for use in the present invention are glycosyltransferasesthat are involved in producing structures containinglacto-N-neotetraose,D-galactosyl-β-1,4-N-acetyl-D-glucosaminyl-β-1,3-D-galactosyl-β-1,4-D-glucose,and the P^(k) blood group trisaccharide sequence,D-galactosyl-β-1,4-D-galactosyl-β-1,4-D-glucose, which have beenidentified in the LOS of the mucosal pathogens Neisseria gonnorhoeae andN. meningitidis (Scholten et al., J. Med. Microbiol. 41: 236-243(1994)). The genes from N. meningitidis and N. gonorrhoeae that encodethe glycosyltransferases involved in the biosynthesis of thesestructures have been identified from N. meningitidis immunotypes L3 andL1 (Jennings et al., Mol. Microbiol. 18: 729-740 (1995)) and the N.gonorrhoeae mutant F62 (Gotshlich, J. Exp. Med. 180: 2181-2190 (1994)).In N. meningitidis, a locus consisting of three genes, lgtA, lgtB and lgE, encodes the glycosyltransferase enzymes required for addition of thelast three of the sugars in the lacto-N-neotetraose chain (Wakarchuk etal., J. Biol. Chem. 271: 19166-73 (1996)). Recently the enzymaticactivity of the lgtB and lgtA gene product was demonstrated, providingthe first direct evidence for their proposed glycosyltransferasefunction (Wakarchuk et al., J. Biol. Chem. 271(45): 28271-276 (1996)).In N. gonorrhoeae, there are two additional genes, lgtD which addsβ-D-GalNAc to the 3 position of the terminal galactose of thelacto-N-neotetraose structure and lgtC which adds a terminal α-D-Gal tothe lactose element of a truncated LOS, thus creating the P^(k) bloodgroup antigen structure (Gotshlich (1994), supra.). In N. meningitidis,a separate immunotype L1 also expresses the P^(k) blood group antigenand has been shown to carry an lgtC gene (Jennings et al., (1995),supra.). Neisseria glycosyltransferases and associated genes are alsodescribed in U.S. Pat. No. 5,545,553 (Gotschlich). Genes forα1,2-fucosyltransferase and α1,3-fucosyltransferase from Helicobacterpylori has also been characterized (Martin et al., J. Biol. Chem. 272:21349-21356 (1997)). Also of use in the present invention are theglycosyltransferases of Campylobacter jejuni (see, for example,http://afmb.cnrs-mrs.fr/˜pedro/CAZY/gtf_(—)42.html).

Sulfotransferases

The invention also provides methods for producing peptides that includesulfated molecules, including, for example sulfated polysaccharides suchas heparin, heparan sulfate, carragenen, and related compounds. Suitablesulfotransferases include, for example, chondroitin-6-sulphotransferase(chicken cDNA described by Fukuta et al., J. Biol. Chem. 270:18575-18580 (1995); GenBank Accession No. D49915), glycosaminoglycanN-acetylglucosamine N-deacetylase/N-sulphotransferase 1 (Dixon et al.,Genomics 26: 239-241 (1995); UL18918), and glycosaminoglycanN-acetylglucosamine N-deacetylase/N-sulphotransferase 2 (murine cDNAdescribed in Orellana et al., J. Biol. Chem. 269: 2270-2276 (1994) andEriksson et al., J. Biol. Chem. 269: 10438-10443 (1994); human cDNAdescribed in GenBank Accession No. U2304).

Cell-Bound Glycosyltransferases

In another embodiment, the enzymes utilized in the method of theinvention are cell-bound glycosyltransferases. Although many solubleglycosyltransferases are known (see, for example, U.S. Pat. No.5,032,519), glycosyltransferases are generally in membrane-bound formwhen associated with cells. Many of the membrane-bound enzymes studiedthus far are considered to be intrinsic proteins; that is, they are notreleased from the membranes by sonication and require detergents forsolubilization. Surface glycosyltransferases have been identified on thesurfaces of vertebrate and invertebrate cells, and it has also beenrecognized that these surface transferases maintain catalytic activityunder physiological conditions. However, the more recognized function ofcell surface glycosyltransferases is for intercellular recognition(Roth, MOLECULAR APPROACHES to SUPRACELLULAR PHENOMENA, 1990).

Methods have been developed to alter the glycosyltransferases expressedby cells. For example, Larsen et al., Proc. Natl. Acad. Sci. USA 86:8227-8231 (1989), report a genetic approach to isolate cloned cDNAsequences that determine expression of cell surface oligosaccharidestructures and their cognate glycosyltransferases. A cDNA librarygenerated from mRNA isolated from a murine cell line known to expressUDP-galactose:.β.-D-galactosyl-1,4-N-acetyl-D-glucosaminideα-1,3-galactosyltransferase was transfected into COS-1 cells. Thetransfected cells were then cultured and assayed for α 1-3galactosyltransferase activity.

Francisco et al., Proc. Natl. Acad. Sci. USA 89: 2713-2717 (1992),disclose a method of anchoring β-lactamase to the external surface ofEscherichia coli. A tripartite fusion consisting of (i) a signalsequence of an outer membrane protein, (ii) a membrane-spanning sectionof an outer membrane protein, and (iii) a complete mature β-lactamasesequence is produced resulting in an active surface bound β-lactamasemolecule. However, the Francisco method is limited only to procaryoticcell systems and as recognized by the authors, requires the completetripartite fusion for proper functioning.

Fusion Proteins

In other exemplary embodiments, the methods of the invention utilizefusion proteins that have more than one enzymatic activity that isinvolved in synthesis of a desired glycopeptide conjugate. The fusionpolypeptides can be composed of, for example, a catalytically activedomain of a glycosyltransferase that is joined to a catalytically activedomain of an accessory enzyme. The accessory enzyme catalytic domaincan, for example, catalyze a step in the formation of a nucleotide sugarthat is a donor for the glycosyltransferase, or catalyze a reactioninvolved in a glycosyltransferase cycle. For example, a polynucleotidethat encodes a glycosyltransferase can be joined, in-frame, to apolynucleotide that encodes an enzyme involved in nucleotide sugarsynthesis. The resulting fusion protein can then catalyze not only thesynthesis of the nucleotide sugar, but also the transfer of the sugarmoiety to the acceptor molecule. The fusion protein can be two or morecycle enzymes linked into one expressible nucleotide sequence. In otherembodiments the fusion protein includes the catalytically active domainsof two or more glycosyltransferases. See, for example, U.S. Pat. No.5,641,668. The modified glycopeptides of the present invention can bereadily designed and manufactured utilizing various suitable fusionproteins (see, for example, PCT Patent Application PCT/CA98/01180, whichwas published as WO 99/31224 on Jun. 24, 1999.)

Immobilized Enzymes

In addition to cell-bound enzymes, the present invention also providesfor the use of enzymes that are immobilized on a solid and/or solublesupport. In an exemplary embodiment, there is provided aglycosyltransferase that is conjugated to a PEG via an intact glycosyllinker according to the methods of the invention. The PEG-linker-enzymeconjugate is optionally attached to solid support. The use of solidsupported enzymes in the methods of the invention simplifies the work upof the reaction mixture and purification of the reaction product, andalso enables the facile recovery of the enzyme. The glycosyltransferaseconjugate is utilized in the methods of the invention. Othercombinations of enzymes and supports will be apparent to those of skillin the art.

Exemplary peptides with which the present invention can be practiced,methods of adding or removing glycosylation sites, and adding orremoving glycosyl structures or substructures are described in detail inWO03/031464 and related U.S. and PCT applications.

The present invention also takes advantage of adding to (or removingfrom) a peptide one or more selected glycosyl residues, after which amodified sugar is conjugated to at least one of the selected glycosylresidues of the peptide. The present embodiment is useful, for example,when it is desired to conjugate the modified sugar to a selectedglycosyl residue that is either not present on a peptide or is notpresent in a desired amount. Thus, prior to coupling a modified sugar toa peptide, the selected glycosyl residue is conjugated to the peptide byenzymatic or chemical coupling. In another embodiment, the glycosylationpattern of a glycopeptide is altered prior to the conjugation of themodified sugar by the removal of a carbohydrate residue from theglycopeptide. See, for example WO 98/31826.

Addition or removal of any carbohydrate moieties present on theglycopeptide is accomplished either chemically or enzymatically. Anexemplary chemical deglycosylation is brought about by exposure of thepolypeptide variant to the compound trifluoromethanesulfonic acid, or anequivalent compound. This treatment results in the cleavage of most orall sugars except the linking sugar (N-acetylglucosamine orN-acetylgalactosamine), while leaving the peptide intact. Chemicaldeglycosylation is described by Hakimuddin et al., Arch. Biochem.Biophys. 259: 52 (1987) and by Edge et al., Anal. Biochem. 118: 131(1981). Enzymatic cleavage of carbohydrate moieties on polypeptidevariants can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al., Meth. Enzymol. 138:350 (1987).

In an exemplary embodiment, the peptide is essentially completelydesialylated with neuraminidase prior to performing glycoconjugation orremodeling steps on the peptide. Following the glycoconjugation orremodeling, the peptide is optionally re-sialylated using asialyltransferase. In an exemplary embodiment, the re-sialylation occursat essentially each (e.g., >80%, preferably greater than 85%, greaterthan 90%, preferably greater than 95% and more preferably greater than96%, 97%, 98% or 99%) terminal saccharyl acceptor in a population ofsialyl acceptors. In a preferred embodiment, the saccharide has asubstantially uniform sialylation pattern (i.e., substantially uniformglycosylation pattern).

Chemical addition of glycosyl moieties is carried out by anyart-recognized method. Enzymatic addition of sugar moieties ispreferably achieved using a modification of the methods set forthherein, substituting native glycosyl units for the modified sugars usedin the invention. Other methods of adding sugar moieties are disclosedin U.S. Pat. Nos. 5,876,980, 6,030,815, 5,728,554, and 5,922,577.

Exemplary attachment points for selected glycosyl residue include, butare not limited to: (a) consensus sites for N-linked glycosylation, andsites for O-linked glycosylation; (b) terminal glycosyl moieties thatare acceptors for a glycosyltransferase; (c) arginine, asparagine andhistidine; (d) free carboxyl groups; (e) free sulfhydryl groups such asthose of cysteine; (f) free hydroxyl groups such as those of serine,threonine, or hydroxyproline; (g) aromatic residues such as those ofphenylalanine, tyrosine, or tryptophan; or (h) the amide group ofglutamine. Exemplary methods of use in the present invention aredescribed in WO 87/05330 published Sep. 11, 1987, and in Aplin andWriston, CRC CRIT. REV. BIOCHEM., pp. 259-306 (1981).

In an exemplary embodiment, the peptide that is modified by a method ofthe invention is a glycopeptide that is produced in mammalian cells(e.g., CHO cells) or in a transgenic animal and thus, contains N- and/orO-linked oligosaccharide chains, which are incompletely sialylated. Theoligosaccharide chains of the glycopeptide lacking a sialic acid andcontaining a terminal galactose residue can be PEGylated, PPGylated orotherwise modified with a modified sialic acid.

Exemplary PEG-sialic acid derivative include:

in which L is a substituted or unsubstituted alkyl or substituted orunsubstituted heteroalkyl linker moiety joining the sialic acid moietyand the PEG moiety, and “n” is 1 or greater; and

in which the index “s” represents an integer from 0 to 20, and “n” is 1or greater.Linker Groups (Cross-Linking Groups)

Preparation of the modified sugar for use in the methods of the presentinvention includes attachment of a modifying group to a sugar residueand forming a stable adduct, which is a substrate for aglycosyltransferase. The sugar and modifying group can be coupled by azero- or higher-order cross-linking agent. Exemplary bifunctionalcompounds which can be used for attaching modifying groups tocarbohydrate moieties include, but are not limited to, bifunctionalpoly(ethylene glycols), polyamides, polyethers, polyesters and the like.General approaches for linking carbohydrates to other molecules areknown in the literature. See, for example, Lee et al., Biochemistry 28:1856 (1989); Bhatia et al., Anal. Biochem. 178: 408 (1989); Janda etal., J. Am. Chem. Soc. 112: 8886 (1990) and Bednarski et al., WO92/18135. In the discussion that follows, the reactive groups aretreated as benign on the sugar moiety of the nascent modified sugar. Thefocus of the discussion is for clarity of illustration. Those of skillin the art will appreciate that the discussion is relevant to reactivegroups on the modifying group as well.

Cleavable Linker Groups

In yet a further embodiment, the linker group is provided with a groupthat can be cleaved to release the modifying group from the sugarresidue. Many cleaveable groups are known in the art. See, for example,Jung et al., Biochem. Biophys. Acta 761: 152-162 (1983); Joshi et al.,J. Biol. Chem. 265: 14518-14525 (1990); Zarling et al., J. Immunol. 124:913-920 (1980); Bouizar et al., Eur. J. Biochem. 155: 141-147 (1986);Park et al., J. Biol. Chem. 261: 205-210 (1986); Browning et al., J.Immunol. 143: 1859-1867 (1989). Moreover a broad range of cleavable,bifunctional (both homo- and hetero-bifunctional) linker groups iscommercially available from suppliers such as Pierce.

Exemplary cleaveable moieties can be cleaved using light, heat orreagents such as thiols, hydroxylamine, bases, periodate and the like.Moreover, certain preferred groups are cleaved in vivo in response tobeing endocytized (e.g., cis-aconityl; see, Shen et al., Biochem.Biophys. Res. Commun. 102: 1048 (1991)). Preferred cleaveable groupscomprise a cleaveable moiety which is a member selected from the groupconsisting of disulfide, ester, imide, carbonate, nitrobenzyl, phenacyland benzoin groups.

Conjugation of Modified Sugars to Peptides

The modified sugars are conjugated to a glycosylated or non-glycosylatedpeptide using an appropriate enzyme to mediate the conjugation.Preferably, the concentrations of the modified donor sugar(s), enzyme(s)and acceptor peptide(s) are selected such that glycosylation proceedsuntil the acceptor is consumed. The considerations discussed below,while set forth in the context of a sialyltransferase, are generallyapplicable to other glycosyltransferase reactions.

A number of methods of using glycosyltransferases to synthesize desiredoligosaccharide structures are known and are generally applicable to theinstant invention. Exemplary methods are described, for instance, WO96/32491, Ito et al., Pure Appl. Chem. 65: 753 (1993), and U.S. Pat.Nos. 5,352,670, 5,374,541, 5,545,553, commonly owned U.S. Pat. Nos.6,399,336, and 6,440,703, and commonly owned published PCT applications,WO 03/031464, WO 04/033651, WO 04/099231, which are incorporated hereinby reference.

The present invention is practiced using a single glycosyltransferase ora combination of glycosyltransferases. For example, one can use acombination of a sialyltransferase and a galactosyltransferase. In thoseembodiments using more than one enzyme, the enzymes and substrates arepreferably combined in an initial reaction mixture, or the enzymes andreagents for a second enzymatic reaction are added to the reactionmedium once the first enzymatic reaction is complete or nearly complete.By conducting two enzymatic reactions in sequence in a single vessel,overall yields are improved over procedures in which an intermediatespecies is isolated. Moreover, cleanup and disposal of extra solventsand by-products is reduced.

In a preferred embodiment, each of the first and second enzyme is aglycosyltransferase. In another preferred embodiment, one enzyme is anendoglycosidase. In an additional preferred embodiment, more than twoenzymes are used to assemble the modified glycoprotein of the invention.The enzymes are used to alter a saccharide structure on the peptide atany point either before or after the addition of the modified sugar tothe peptide.

In another embodiment, the method makes use of one or more exo- orendoglycosidase. The glycosidase is typically a mutant, which isengineered to form glycosyl bonds rather than cleave them. The mutantglycanase typically includes a substitution of an amino acid residue foran active site acidic amino acid residue. For example, when theendoglycanase is endo-H, the substituted active site residues willtypically be Asp at position 130, Glu at position 132 or a combinationthereof. The amino acids are generally replaced with serine, alanine,asparagine, or glutamine.

The mutant enzyme catalyzes the reaction, usually by a synthesis stepthat is analogous to the reverse reaction of the endoglycanasehydrolysis step. In these embodiments, the glycosyl donor molecule(e.g., a desired oligo- or mono-saccharide structure) contains a leavinggroup and the reaction proceeds with the addition of the donor moleculeto a GlcNAc residue on the protein. For example, the leaving group canbe a halogen, such as fluoride. In other embodiments, the leaving groupis a Asn, or a Asn-peptide moiety. In yet further embodiments, theGlcNAc residue on the glycosyl donor molecule is modified. For example,the GlcNAc residue may comprise a 1,2 oxazoline moiety.

In a preferred embodiment, each of the enzymes utilized to produce aconjugate of the invention are present in a catalytic amount. Thecatalytic amount of a particular enzyme varies according to theconcentration of that enzyme's substrate as well as to reactionconditions such as temperature, time and pH value. Means for determiningthe catalytic amount for a given enzyme under preselected substrateconcentrations and reaction conditions are well known to those of skillin the art.

The temperature at which an above process is carried out can range fromjust above freezing to the temperature at which the most sensitiveenzyme denatures. Preferred temperature ranges are about 0° C. to about55° C., and more preferably about 20° C. to about 30° C. In anotherexemplary embodiment, one or more components of the present method areconducted at an elevated temperature using a thermophilic enzyme.

The reaction mixture is maintained for a period of time sufficient forthe acceptor to be glycosylated, thereby forming the desired conjugate.Some of the conjugate can often be detected after a few hours, withrecoverable amounts usually being obtained within 24 hours or less.Those of skill in the art understand that the rate of reaction isdependent on a number of variable factors (e.g., enzyme concentration,donor concentration, acceptor concentration, temperature, solventvolume), which are optimized for a selected system.

The present invention also provides for the industrial-scale productionof modified peptides. As used herein, an industrial scale generallyproduces at least 1 gram of finished, purified conjugate.

In the discussion that follows, the invention is exemplified by theconjugation of modified sialic acid moieties to a glycosylated peptide.The exemplary modified sialic acid is labeled with m-PEG. The focus ofthe following discussion on the use of PEG-modified sialic acid andglycosylated peptides is for clarity of illustration and is not intendedto imply that the invention is limited to the conjugation of these twopartners. One of skill understands that the discussion is generallyapplicable to the additions of modified glycosyl moieties other thansialic acid. Moreover, the discussion is equally applicable to themodification of a glycosyl unit with agents other than m-PEG includingother PEG moieties, therapeutic moieties, and biomolecules.

An enzymatic approach can be used for the selective introduction ofm-PEGylated or m-PPGylated carbohydrates onto a peptide or glycopeptide.The method utilizes modified sugars containing PEG, PPG, or a maskedreactive functional group, and is combined with the appropriateglycosyltransferase or glycosynthase. By selecting theglycosyltransferase that will make the desired carbohydrate linkage andutilizing the modified sugar as the donor substrate, the PEG or PPG canbe introduced directly onto the peptide backbone, onto existing sugarresidues of a glycopeptide or onto sugar residues that have been addedto a peptide.

In an exemplary embodiment, an acceptor for a sialyltransferase ispresent on the peptide to be modified either as a naturally occurringstructure or is placed there recombinantly, enzymatically or chemically.Suitable acceptors, include, for example, galactosyl acceptors such asGalβ1,4GlcNAc, Galβ1,4GalNAc, Galβ1,3GalNAc, lacto-N-tetraose,Galβ1,3GlcNAc, GalNAc, Galβ1,3GalNAc, Galβ11,6GlcNAc, Galβ1,4Glc(lactose), and other acceptors known to those of skill in the art (see,e.g., Paulson et al., J. Biol. Chem. 253: 5617-5624 (1978)). Exemplarysialytransferases are set forth herein.

In one embodiment, an acceptor for the sialyltransferase is present onthe glycopeptide to be modified upon in vivo synthesis of theglycopeptide. Such glycopeptides can be sialylated using the claimedmethods without prior modification of the glycosylation pattern of theglycopeptide. Alternatively, the methods of the invention can be used tosialylate a peptide that does not include a suitable acceptor; one firstmodifies the peptide to include an acceptor by methods known to those ofskill in the art. In an exemplary embodiment, a GalNAc residue is addedby the action of a GalNAc transferase.

In an exemplary embodiment, the galactosyl acceptor is assembled byattaching a galactose residue to an appropriate acceptor linked to thepeptide, e.g., a GlcNAc. The method includes incubating the peptide tobe modified with a reaction mixture that contains a suitable amount of agalactosyltransferase (e.g., Galβ1,3 or Galβ1,4), and a suitablegalactosyl donor (e.g., UDP-galactose). The reaction is allowed toproceed substantially to completion or, alternatively, the reaction isterminated when a preselected amount of the galactose residue is added.Other methods of assembling a selected saccharide acceptor will beapparent to those of skill in the art.

In yet another embodiment, glycopeptide-linked oligosaccharides arefirst “trimmed,” either in whole or in part, to expose either anacceptor for the sialyltransferase or a moiety to which one or moreappropriate residues can be added to obtain a suitable acceptor. Enzymessuch as glycosyltransferases and endoglycosidases (see, for example U.S.Pat. No. 5,716,812) are useful for the attaching and trimming reactions.In another embodiment of this method, the sialic acid moieties of thepeptide are essentially completely removed (e.g., at least 90, at least95 or at least 99%), exposing an acceptor for a modified sialic acid.

In the discussion that follows, the method of the invention isexemplified by the use of modified sugars having a PEG moiety attachedthereto. The focus of the discussion is for clarity of illustration.Those of skill will appreciate that the discussion is equally relevantto those embodiments in which the modified sugar bears a therapeuticmoiety, biomolecule or the like.

In an exemplary embodiment of the invention in which a carbohydrateresidue is “trimmed” prior to the addition of the modified sugar, highmannose is trimmed back to the first generation biantennary structure. Amodified sugar bearing a PEG moiety is conjugated to one or more of thesugar residues exposed by the “trimming back.” In one example, a PEGmoiety is added via a GlcNAc conjugated to the PEG moiety. The modifiedGlcNAc is attached to one or both of the terminal mannose residues ofthe biantennary structure. Alternatively, an unmodified GlcNAc can beadded to one or both of the termini of the branched species.

In another exemplary embodiment, a PEG moiety is added to one or both ofthe terminal mannose residues of the biantennary structure via amodified sugar having a galactose residue, which is conjugated to aGlcNAc residue added onto the terminal mannose residues. Alternatively,an unmodified Gal can be added to one or both terminal GlcNAc residues.

In yet a further example, a PEG moiety is added onto a Gal residue usinga modified sialic acid such as those discussed above.

In another exemplary embodiment, an O-linked glycosyl residue is“trimmed back” to the GalNAc attached to the amino acid. In one example,a water-soluble polymer is added via a Gal modified with the polymer.Alternatively, an unmodified Gal is added to the GalNAc, followed by aGal with an attached water-soluble polymer. In yet another embodiment,one or more unmodified Gal residue is added to the GalNAc, followed by asialic acid moiety modified with a water-soluble polymer.

A high mannose structure can also be trimmed back to the elementarytri-mannosyl core.

In a further exemplary embodiment, high mannose is “trimmed back” to theGlcNAc to which the first mannose is attached. The GlcNAc is conjugatedto a Gal residue bearing a PEG moiety. Alternatively, an unmodified Galis added to the GlcNAc, followed by the addition of a sialic acidmodified with a water-soluble sugar. In yet a further example, theterminal GlcNAc is conjugated with Gal and the GlcNAc is subsequentlyfucosylated with a modified fucose bearing a PEG moiety.

High mannose may also be trimmed back to the first GlcNAc attached tothe Asn of the peptide. In one example, the GlcNAc of theGlcNAc-(Fuc)_(a) residue is conjugated with a GlcNAc bearing a watersoluble polymer. In another example, the GlcNAc of the GlcNAc-(Fuc)_(a)residue is modified with Gal, which bears a water soluble polymer. In astill further embodiment, the GlcNAc is modified with Gal, followed byconjugation to the Gal of a sialic acid modified with a PEG moiety.

Other exemplary embodiments are set forth in commonly owned U.S. Patentapplication Publications: 20040132640; 20040063911; 20040137557; U.S.patent application Ser. Nos. 10/369,979; 10/410,913; 10/360,770;10/410,945 and PCT/US02/32263 each of which is incorporated herein byreference.

The Examples set forth above provide an illustration of the power of themethods set forth herein. Using the methods of the invention, it ispossible to “trim back” and build up a carbohydrate residue ofsubstantially any desired structure. The modified sugar can be added tothe termini of the carbohydrate moiety as set forth above, or it can beintermediate between the peptide core and the terminus of thecarbohydrate.

Glycosylation by Recombinant Methods

Glycosylation of a mutant human growth hormone may also be accomplishedintracellularly by recombinant means. A polynucleotide sequence encodinga mutant human growth hormone, which comprises at least one newlyintroduced N- or O-linked glycosylation site, may be transfected into asuitable host cell line, e.g., a eukaryotic cell line derived fromyeast, insect, or mammalian origin. The mutant human growth hormonerecombinantly produced from such a cell line is glycosylated by the hostcell glycosylation machinery.

In a selected embodiment, a hGH peptide, expressed in insect cells, isremodeled such that glycans on the remodeled glycopeptide include aGlcNAc-Gal glycosyl residue. The addition of GlcNAc and Gal can occur asseparate reactions or as a single reaction in a single vessel. In thisexample, GlcNAc-transferase I and Gal-transferase I are used. Themodified sialyl moiety is added using ST3Gal-Ill.

In another embodiment, the addition of GlcNAc, Gal and modified Sia canalso occur in a single reaction vessel, using the enzymes set forthabove. Each of the enzymatic remodeling and glycoPEGylation steps arecarried out individually.

When the peptide is expressed in mammalian cells, different methods areof use. In one embodiment, the peptide is conjugated without need forremodeling prior to conjugation by contacting the peptide with asialyltransferase that transfers the modified sialic acid directly ontoa sialic acid on the peptide forming Sia-Sia-L-R¹, or exchanges a sialicacid on the peptide for the modified sialic acid, forming Sia-L-R¹. Anexemplary enzyme of use in this method is CST-II. Other enzymes that addsialic acid to sialic acid are known to those of skill in the art andexamples of such enzymes are set forth the figures appended hereto.

In yet another method of preparing the conjugates of the invention, thepeptide expressed in a mammalian system is desialylated using asialidase. The exposed Gal residue is sialylated with a modified sialicacid using a sialyltransferase specific for O-linked glycans, providingan hGH peptide with an O-linked modified glycan. The desialylated,modified hGH peptide is optionally partially or fully re-sialylated byusing a sialyltransferase such as ST3GalIII.

In another aspect, the invention provides a method of making a PEGylatedhGH of the invention. The method includes: (a) contacting a substratehGH peptide comprising a glycosyl group selected from:

with a sugar donor and an enzyme for which the sugar donor is asubstrate under conditions appropriate to transfer a sugar moiety fromthe sugar donor to the hGH peptide. In an exemplary embodiment, thesugar moiety is a modified sugar moiety. In another exemplaryembodiment, the modified sugar moiety is a modified sialic acid residuewith a water-soluble polymer (e.g., PEG) covalently attached thereto. Anexemplary PEG-sialic acid sugar donor has the formula:

A preferred enzyme transfers PEG-sialic acid from said donor onto amember selected from the GalNAc, Gal and the Sia of a glycosyl group,under conditions appropriate for the transfer. An exemplary modifiedsialic acid donor is CMP-sialic acid modified, through a linker moiety,with a polymer, e.g., a straight chain or branched poly(ethylene glycol)moiety. As discussed herein, the peptide is optionally glycosylated withGalNAc and/or Gal and/or Sia (“Remodeled”) prior to attaching themodified sugar. The remodeling steps can occur in sequence in the samevessel without purification of the glycosylated peptide between steps.Alternatively, following one or more remodeling step, the glycosylatedpeptide can be purified prior to submitting it to the next glycosylationor glycPEGylation step.

As illustrated in the examples and discussed further below, placement ofan acceptor moiety for the PEG-sugar is accomplished in any desirednumber of steps. For example, in one embodiment, the addition of GalNActo the peptide can be followed by a second step in which the PEG-sugaris conjugated to the GalNAc in the same reaction vessel. Alternatively,these two steps can be carried out in a single vessel approximatelysimultaneously.

In a further exemplary embodiment, the hGH peptide is expressed in anappropriate expression system prior to being glycopegylated orremodeled. Exemplary expression systems include Sf-9/baculovirus andChinese Hamster Ovary (CHO) cells.

In another exemplary embodiment, the invention provides methods offorming a conjugate of hGH such as those set forth herein in which thehGH in the conjugate is essentially unoxidized. Oxidation of methionineresidues of PEG-hGH can be detected by N-terminal sequencing and peptidemapping. Oxidation or its absence can be confirmed using RP-HPLC. Forexample, using RP-HPLC, a peak in addition the major PEG-hGH peak wasdetected, which represents a PEG-hGH species in which methionine isoxidized (Met-Ox). For hGH this peak has been identified asMet127/Met138 oxidation, eluting 0.2 min before the main peak.Additionally, a small peak eluting approximately 3 min before the mainpeak as Met122 oxidation has been identified. Met1 oxidation wasdetected by RP-HPLC using the 60° C. method, but coelutes with the mainpeak. This N-terminal methionine oxidation is detected by peptidemapping and is referred to as G1-Ox.

Thus, in an exemplary embodiment, the invention provides a population ofhGH conjugates, as described herein, in which less than 10%, preferablyless than 5%, more preferably less than 1%, more preferably less than0.5%, still more preferably less than 0.1%, preferably less than 0.05%,more preferably less than 0.01%, even more preferably less than 0.005%and still more preferably less than 0.001% of the members of thepopulation include a methionine residue selected from Met127, Met138,Met 122, N-terminal Met and combinations thereof which is oxidized.

In an exemplary method according to the invention, the enzymaticconjugation of the modified sugar to the peptide is performed underconditions that prevent or retard the oxidation of methionine residuesof the peptide. In an exemplary embodiment, the reaction mixtureincludes added methionine. Exemplary methods of the invention use up toabout 20 mM methionine in the conjugation reaction mixture.

Purification of Glycosylated Mutant hGH

The products produced by the above processes can be used withoutpurification. However, it is usually preferred to recover the productand one or more of the intermediates, e.g., nucleotide sugars, branchedand linear PEG species, modified sugars and modified nucleotide sugars.Standard, well known techniques for recovery of glycosylated saccharidessuch as thin or thick layer chromatography, column chromatography, ionexchange chromatography, or membrane filtration can be used. It ispreferred to use membrane filtration, more preferably utilizing areverse osmotic membrane, or one or more column chromatographictechniques for the recovery as is discussed hereinafter and in theliterature cited herein. For instance, membrane filtration wherein themembranes have molecular weight cutoff of about 3000 to about 10,000 canbe used to remove proteins such as glycosyl transferases. Nanofiltrationor reverse osmosis can then be used to remove salts and/or purify theproduct saccharides (see, e.g., WO 98/15581). Nanofilter membranes are aclass of reverse osmosis membranes that pass monovalent salts but retainpolyvalent salts and uncharged solutes larger than about 100 to about2,000 Daltons, depending upon the membrane used. Thus, in a typicalapplication, saccharides prepared by the methods of the presentinvention will be retained in the membrane and contaminating salts willpass through.

If the glycosylated mutant human growth hormone is producedintracellularly, as a first step, the particulate debris, either hostcells or lysed fragments, is removed. Following glycoPEGylation, thePEGylated peptide is purified by art-recognized methods, for example, bycentrifugation or ultrafiltration; optionally, the protein may beconcentrated with a commercially available protein concentration filter,followed by separating the polypeptide variant from other impurities byone or more steps selected from immunoaffinity chromatography,ion-exchange column fractionation (e.g., on diethylaminoethyl (DEAE) ormatrices containing carboxymethyl or sulfopropyl groups), chromatographyon Blue-Sepharose, CM Blue-Sepharose, MONO-Q, MONO-S, lentillectin-Sepharose, WGA-Sepharose, Con A-Sepharose, Ether Toyopearl, ButylToyopearl, Phenyl Toyopearl, SP-Sepharose, or protein A Sepharose,SDS-PAGE chromatography, silica chromatography, chromatofocusing,reverse phase HPLC (e.g., silica gel with appended aliphatic groups),gel filtration using, e.g., Sephadex molecular sieve or size-exclusionchromatography, chromatography on columns that selectively bind thepolypeptide, and ethanol or ammonium sulfate precipitation.

Modified glycopeptides produced in culture are usually isolated byinitial extraction from cells, cell lysate, culture media, etc.,followed by one or more concentration, salting-out, aqueousion-exchange, or size-exclusion chromatography steps. Additionally, themodified glycoprotein may be purified by affinity chromatography.Finally, HPLC may be employed for final purification steps.

A protease inhibitor, e.g., methylsulfonylfluoride (PMSF) may beincluded in any of the foregoing steps to inhibit proteolysis andantibiotics or preservatives may be included to prevent the growth ofadventitious contaminants.

In another embodiment, supernatants from systems that produce themodified human growth hormone of the invention are first concentratedusing a commercially available protein concentration filter, forexample, an Amicon or Millipore Pellicon ultrafiltration unit. Followingthe concentration step, the concentrate may be applied to a suitablepurification matrix. For example, a suitable affinity matrix maycomprise a ligand for the peptide, a lectin or antibody molecule boundto a suitable support. Alternatively, an anion-exchange resin may beemployed, for example, a matrix or substrate having pendant DEAE groups.Suitable matrices include acrylamide, agarose, dextran, cellulose, orother types commonly employed in protein purification. Alternatively, acation-exchange step may be employed. Suitable cation exchangers includevarious insoluble matrices comprising sulfopropyl or carboxymethylgroups. Sulfopropyl groups are particularly preferred.

Other methods of use in purification include size exclusionchromatography (SEC), hydroxyapatite chromatography, hydrophobicinteraction chromatography and chromatography on Blue Sepharose. Theseand other useful methods are illustrated in co-assigned U.S. ProvisionalPatent No. (Attorney Docket No. 40853-01-5168-P1, filed May 6, 2005).

One or more RP-HPLC steps employing hydrophobic RP-HPLC media, e.g.,silica gel having pendant methyl or other aliphatic groups, may beemployed to further purify a polypeptide conjugate composition. Some orall of the foregoing purification steps, in various combinations, canalso be employed to provide a homogeneous or essentially homogeneousmodified glycoprotein.

The modified glycopeptide of the invention resulting from a large-scalefermentation may be purified by standard methods analogous to thosedisclosed by Urdal et al., J. Chromatog. 296: 171 (1984). This referencedescribes two sequential, RP-HPLC steps for purification of recombinanthuman IL-2 on a preparative HPLC column. Alternatively, techniques suchas affinity chromatography may be utilized to purify the modifiedglycoprotein. For instance, chemically PEGylated mutant hGH products ofthe invention may be fractionated using standard chromatographictechniques known in the art to obtain a more homogenous productcontaining specific numbers of PEG or fractions of PEG (e.g. 1-3, 2-4,etc.).

In an exemplary embodiment, the purification is accomplished by themethods set forth in commonly owned, co-assigned U.S. Provisional PatentNo. 60/665,588, filed Mar. 24, 2005.

In another exemplary embodiment, the purification is effected by SPHPchromatography using an appropriate buffer as an eluent. Exemplarybuffers include citrate and acetate buffers, with citrate presentlypreferred.

In a further exemplary embodiment, a phosphate salt, e.g, sodiumphosphate is added to the enzymatic conjugation reaction mixture. Thereaction mixture is centrifuged and the resulting mixture is purified bySPHP. In this embodiment, free methionine, which is not covalentlyattached to the hGH peptide, is either present or absent during thepurification step.

An exemplary purification process, as set forth above, results in theisolation of a population of hGH conjugates, as described herein, inwhich less than 10%, preferably less than 5%, more preferably less than1%, more preferably less than 0.5%, still more preferably less than0.1%, preferably less than 0.05%, more preferably less than 0.01%, evenmore preferably less than 0.005% and still more preferably less than0.001% of the members of the population include a methionine residueselected from Met127, Met138, Met 122, N-terminal Met and combinationsthereof which is oxidized.

In yet another exemplary embodiment, the purified hGH conjugatecomposition includes a population of hGH peptides in which less than10%, preferably less than 5%, more preferably less than 1%, morepreferably less than 0.5%, still more preferably less than 0.1%,preferably less than 0.05%, more preferably less than 0.01%, even morepreferably less than 0.005% and still more preferably less than 0.001%of the population of peptides is associated in a peptide aggregate asdetermined by size-exclusion chromatography.

Protease Resistant Mutant hGH

The present invention also provides protease resistant hGH mutants. Asused herein, the terms “protease resistant” and “proteolysis resistant”refer to hGH peptides (e.g., mutants) that enhance the stability of themutant hGH peptide relative to the wild type hGH peptide in the presenceof a protease. In a preferred embodiment, the protease recognition siteof the wild type is completely inactivated in the mutant and is not asite of cleavage by the protease that cleaved the corresponding site inthe wild type. In exemplary embodiments, the rate of cleavage is reducedby about 30%, more preferably by about 60%, and even more preferably byabout 90%.

In an exemplary embodiment, protease resistance is imbued by mutation ofone or more amino acids within a protease recognition site. In anotherexemplary embodiment, protease resistance is imparted by the presence ofone or more water-soluble polymer moieties covalently bound to thepeptide proximate the protease recognition site. The water-solublepeptide can be placed by reaction with an activated water-solublepolymer (e.g., NHS-ester of PEG) or enzymatically (e.g., byglycoPEGylation). Those of skill will appreciate that a combination ofmutation and conjugation of a water-soluble polymer can be used.

In an exemplary embodiment, chemical PEGylation of hGH prior to and/orafter glycoPEGylation is used to achieve the protease resistant hGH ofthe present invention. These two types of peptide PEGylation can beperformed in any combinations and order. Chemical PEGylation, ascontemplated in the present invention, involves the binding of PEG, orother blocking group, i.e. a group inhibiting proteolysis, either in ornear various protease recognition sites, thereby allowing the PEG, orother group, to serve essentially as a protease inhibitor. Peptideschemically PEGylated in this manner show enhanced resistance to proteasedegradation.

Methods for chemically PEGylating peptides are widely-known in the art(e.g. see Background section). Chemical PEGylation is performed with anyacceptable PEGylation reagent. Exemplary standard activated PEG groupsknown in the art can be used for this process (see, Shearwater Polymers,Nektar Therapeutics catalogs). Moreover, the linear and branched PEGspecies set forth herein are similarly of use in practicing the presentmethods. It will be apparent to those of skill that the PEG moleculesset forth herein are readily conversted to useful activated derivativesby methods known in the art.

In preferred embodiments, the molecular weights of PEG to be usedinclude 500MW, 2 kDa, 5 kDa, 10 kDa, 20 kDa, 30 kDa, and 40 kDa, etc.Other acceptable branched and linear PEG structures are discussed in thepreceding sections.

It should be noted that protease inhibiting groups other than PEG,including other water-soluble polymers, water-insoluble polymers,therapeutic moieties, and biomolecules, are also useful in practicingthe present invention.

Functional Assays for the Mutant hGH

Following the production and, preferably, purification of a glycosylatedmutant human growth hormone, the biological functions of theglycoprotein are tested using several methods known in the art. Thefunctional assays are based on various characteristics of human growthhormone, such as its specific binding to human growth hormone receptor,activation of the hGH receptor, and its activity in promoting cellgrowth. In each assay, wild-type human growth hormone is included as apositive control.

A radioreceptor binding assay can be carried out to measure the bindingbetween a radio-labeled hGH receptor and a mutant human growth hormoneof the present invention. Detailed description for such an assay can befound in the literature, e.g., Tsushima et al., J Clin. Endocrinol.Metab., 37: 334-337 (1973); Chin et al., Endocr. Meta. 37: 334 (1973);and U.S. Pat. Nos. 4,871,835, 5,079,230.

The ability of a mutant human growth hormone to promote cell growth isassessed by methods such as the tibia test (Parlow et al., Endocrinology77: 1126(1965); U.S. Pat. No. 4,871,835). Briefly, rats arehypophysectomized at 28-30 days of age and kept for 10-14 days withouttreatment. Human growth hormone mutants derived from recombinant sourceis then given to the rats by daily subcutaneous injections. The animalsare sacrificed on the sixth day, their foreleg knee bones taken out andthe width of the epiphyseal plates measured. The weight of these rats atthe start of the experiment and before being sacrificed is alsomonitored and compared among different groups receiving daily injectionsof the mutant human growth hormone at different concentrations.

Furthermore, the biological activity of a mutant human growth hormonecan be demonstrated in its ability to cause hGH-dependent tyrosinephosphorylation in 14-9 cells, which are derived from a clone of humanlymphoblastoma and express human growth hormone receptor on the cellsurface. Other cell types such as MB-2 cells may also be suitable forhGH functional assays. The level of tyrosine phosphorylation of cellularproteins upon exposure to the mutant human growth hormone is shown by amonoclonal antibody against phosphorylated tyrosine, as described bySilva et al., Endocrinology, 132: 101 (1993) and U.S. Pat. No.6,238,915.

Pharmaceutical Composition and Administration

The glycosylated mutant human growth hormone having desiredoligosaccharide determinants described above can be used as therapeuticsfor treating a variety of diseases and conditions related to deficiencyin growth hormone. Growth-related conditions that can be treated withthe mutant human growth hormone of the present invention include:dwarfism, short-stature in children and adults, cachexia/muscle wasting,general muscular atrophy, and sex chromosome abnormality (e.g., Turner'sSyndrome). Other conditions may be treated using the mutant hGH of thepresent invention include: short-bowel syndrome, lipodystrophy,osteoporosis, uraemaia, burns, female infertility, bone regeneration,general diabetes, type II diabetes, osteo-arthritis, chronic obstructivepulmonary disease (COPD), and insomnia. The mutant hGH of the inventionmay also be used to promote various healing processes, e.g., generaltissue regeneration, bone regeneration, and wound healing, or as avaccine adjunct. Thus, the present invention also providespharmaceutical compositions comprising an effective amount ofglycosylated mutant human growth hormone, which is produced according tothe methods described above.

In some embodiments of the present invention, the pharmaceuticalcomposition includes a pharmaceutically acceptable diluent and acovalent conjugate between a non-naturally-occurring, PEG moiety,therapeutic moiety or biomolecule and a glycosylated or non-glycosylatedpeptide. The polymer, therapeutic moiety or biomolecule is conjugated tothe peptide via an intact glycosyl linking group interposed between andcovalently linked to both the peptide and the polymer, therapeuticmoiety or biomolecule.

Pharmaceutical compositions of the invention are suitable for use in avariety of drug delivery systems. Suitable formulations for use in thepresent invention are found in Remington's Pharmaceutical Sciences, MacePublishing Company, Philadelphia, Pa., 17th ed. (1985). For a briefreview of methods for drug delivery, see, Langer, Science 249: 1527-1533(1990).

The pharmaceutical compositions may be formulated for any appropriatemanner of administration, including for example, topical, oral, nasal,intravenous, intracranial, intraperitoneal, subcutaneous orintramuscular administration. For parenteral administration, such assubcutaneous injection, the carrier preferably comprises water, saline,alcohol, a fat, a wax or a buffer. For oral administration, any of theabove carriers or a solid carrier, such as mannitol, lactose, starch,magnesium stearate, sodium saccharine, talcum, cellulose, glucose,sucrose, and magnesium carbonate, may be employed. Biodegradablemicrospheres (e.g., polylactate polyglycolate) may also be employed ascarriers for the pharmaceutical compositions of this invention. Suitablebiodegradable microspheres are disclosed, for example, in U.S. Pat. Nos.4,897,268 and 5,075,109.

Commonly, the pharmaceutical compositions are administered parenterally,e.g., subcutaneously or intravenously. Thus, the invention providescompositions for parenteral administration that include the compounddissolved or suspended in an acceptable carrier, preferably an aqueouscarrier, e.g., water, buffered water, saline, PBS and the like. Thecompositions may also contain detergents such as Tween 20 and Tween 80;stabilizers such as mannitol, sorbitol, sucrose, and trehalose; andpreservatives such as EDTA and m-cresol. The compositions may containpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, wetting agents, detergents and thelike.

In some embodiments the glycopeptides of the invention can beincorporated into liposomes formed from standard vesicle-forming lipids.A variety of methods are available for preparing liposomes, as describedin, e.g., Szoka et al., Ann. Rev. Biophys. Bioeng. 9: 467 (1980), U.S.Pat. Nos. 4,235,871, 4,501,728 and 4,837,028. The targeting of liposomesusing a variety of targeting agents (e.g., the sialyl galactosides ofthe invention) is well known in the art (see, e.g., U.S. Pat. Nos.4,957,773 and 4,603,044).

Standard methods for coupling targeting agents to liposomes can be used.These methods generally involve incorporation into liposomes of lipidcomponents, such as phosphatidylethanolamine, which can be activated forattachment of targeting agents, or derivatized lipophilic compounds,such as lipid-derivatized glycopeptides of the invention.

Targeting mechanisms generally require that the targeting agents bepositioned on the surface of the liposome in such a manner that thetarget moieties are available for interaction with the target, forexample, a cell surface receptor. The carbohydrates of the invention maybe attached to a lipid molecule before the liposome is formed usingmethods known to those of skill in the art (e.g., alkylation oracylation of a hydroxyl group present on the carbohydrate with a longchain alkyl halide or with a fatty acid, respectively).

Alternatively, the liposome may be fashioned in such a way that aconnector portion is first incorporated into the membrane at the time offorming the membrane. The connector portion must have a lipophilicportion, which is firmly embedded and anchored in the membrane. It mustalso have a reactive portion, which is chemically available on theaqueous surface of the liposome. The reactive portion is selected sothat it will be chemically suitable to form a stable chemical bond withthe targeting agent or carbohydrate, which is added later. In some casesit is possible to attach the target agent to the connector moleculedirectly, but in most instances it is more suitable to use a thirdmolecule to act as a chemical bridge, thus linking the connectormolecule which is in the membrane with the target agent or carbohydratewhich is extended, three dimensionally, off of the vesicle surface.

The compounds prepared by the methods of the invention may also find useas diagnostic reagents. For example, labeled compounds can be used tolocate areas of inflammation or tumor metastasis in a patient suspectedof having an inflammation. For this use, the compounds can be labeledwith ¹²⁵I, ¹⁴C, or tritium.

The active ingredient used in the pharmaceutical compositions of thepresent invention is glycopegylated hGH and its derivatives having thebiological properties of stimulating granulocyte production. Preferably,the hGH composition of the present invention is administeredparenterally (e.g. IV, IM, SC or IP). Effective dosages are expected tovary considerably depending on the condition being treated and the routeof administration but are expected to be in the range of about 0.1 (˜7U)to 100 (˜7000U) μg/kg body weight of the active material. Preferabledoses for treatment of anemic conditions are about 50 to about 300Units/kg three times a week. Because the present invention provides ahGH with an enhanced in vivo residence time, the stated dosages areoptionally lowered when a composition of the invention is administered.

Preparative methods for species of use in preparing the compositions ofthe invention are generally set forth in various patent publications,e.g., US 20040137557; WO 04/083258; and WO 04/033651. The followingexamples are provided to illustrate the conjugates, and methods and ofthe present invention, but not to limit the claimed invention.

In an exemplary embodiment, the present invention provides apharmaceutical formulation that includes a population of hGH conjugates,such as described herein, in combination with a pharmaceuticallyacceptable diluent. A preferred formulation of the invention includes abuffer, a detergent, and a polyol.

An exemplary formulation includes the peptide conjugate in an amountfrom about 1 mg/mL to about 100 mg/mL, preferably from about 5 mg/mL toabout 75 mg/mL, and more preferably from about 10 mg/mL to about 50mg/mL.

An exemplary formulation includes a buffer at a concentration of about 1mM to about 100 mM, preferably from about 5 mM to about 75 mM, and morepreferably from about 10 mM to about 50 mM.

In an exemplary formulation, the detergent is present in an amount fromabout 0.00001% to about 10%, preferably from about 0.00005% to about 1%,more preferably from about 0.0001% to about 0.1%, more preferably fromabout 0.0005% to about 0.005%, and even more preferably from about0.001% to about 0.01%.

In an exemplary formulation, the polyol is present in an amount of about1 mg/mL to 100 mg/mL, preferably from about 10 mg/mL to about 75 mg/mL,more preferably from about 15 mg/mL to about 50 mg/mL.

In an exemplary embodiment, the pH of the formulation is from about 3 toabout 7.5, preferably from about 4 to about 6.5 and more preferably fromabout 5 to about 6. Whatever the structure of the peptide conjugate, itis generally preferred that it be formulated at a pH that is within arange of about 0.5 pH units of the pI of the peptide.

In an exemplary embodiment, the detergent is Tween, e.g., Tween 20. In afurther exemplary embodiment the polyol is sorbitol. In anotherembodiment, the buffer is sodium acetate.

An exemplary formulation of the invention includes hGH conjugate (2mg/mL) in a mixture with 10 mM NaOAc, 0.003% Tween 20, and 50 mg/mL ofsorbitol at pH 4.0.

These compositions may be sterilized by conventional sterilizationtechniques, or may be sterile filtered. The resulting aqueous solutionsmay be packaged for use as is, or lyophilized, the lyophilizedpreparation being combined with a sterile aqueous carrier prior toadministration. The pH of the preparations typically will be between 3and 11, more preferably from 5 to 9, and most preferably from 7 and 8.

The compositions containing the glycosylated mutant human growth hormonecan be administered for prophylactic and/or therapeutic treatments. Intherapeutic applications, compositions are administered to a patientalready suffering from a disease or condition related to growth hormonedeficiency, in an amount sufficient to cure or at least partially arrestthe symptoms of the disease and its complications. An amount adequate toaccomplish this is defined as a “therapeutically effective dose.”Amounts effective for this use will depend on the severity of thedisease or condition and the weight and general state of the patient,but generally range from about 0.1 mg to about 2,000 mg of glycosylatedmutant human growth hormone per day for a 70 kg patient, with dosages offrom about 5 mg to about 200 mg of the compounds per day being morecommonly used.

In prophylactic applications, compositions containing the glycosylatedmutant human growth hormone of the invention are administered to apatient susceptible to or otherwise at risk of a particular disease.Such an amount is defined to be a “prophylactically effective dose.” Inthis use, the precise amounts again depend on the patient's state ofhealth and weight, but generally range from about 0.1 mg to about 1,000mg per 70 kilogram patient, more commonly from about 5 mg to about 200mg per 70 kg of body weight.

Single or multiple administrations of the compositions can be carriedout with dose levels and pattern being selected by the treatingphysician. In any event, the pharmaceutical formulations should providea quantity of the glycosylated mutant human growth hormone of thisinvention sufficient to effectively treat the patient.

EXAMPLES

The following examples are provided by way of illustration only and notby way of limitation. Those of skill in the art will readily recognize avariety of non-critical parameters that could be changed or modified toyield essentially similar results.

Example 1

Human growth hormone occurs in a variety of different isoforms anddifferent amino acid sequences. The two best characterized forms includeplacental derived hGH, which is also known as GH-V (PDB P01242) andpituitary derived hGH, which is also known as somatotropin orGH-N(P01241); see FIG. 1. The pituitary derived hGH is not glycosylatedand is produced in Escherichia coli as a therapeutic. The placentalderived hGH (GH-V) has one N-glycosylation site at amino acid 140 (seeTable 4 and FIG. 1, see arrow). TABLE 4 Human Growth Hormone (GH-V),Placenta Derived; P01242 (SEQ ID NO:2)fptiplsrlfdnamlrarrlyqlaydtyqefeeayilkeqkysflqnpqtslcfsesiptpsnrvktqqksnlellrisllliqswlepvqllrsvfanslvygasdsnvyrhlkdleegiqtlmwrledgsprtgqifnqsyskfdtkshnddallknygllycfrkdmdkvetflrivqcrsvegscgf            ↑

The pituitary derived hGH (GH-N) can be modified at amino acid position140 to introduce an N-linked glycosylation site by mutating thenucleotide sequence encoding this polypeptide so that instead ofencoding the wild-type lysine (abbreviated as “k” at amino acid 140 ofthe GH-N polypeptide sequence in Table 5 and FIG. 1, see arrow), thenucleotide sequence will encode an asparagine (abbreviated “n”) at aminoacid position 140 of GH-N (see also FIG. 2). TABLE 5 Human GrowthHormone (GH-N), Pituitary Derived; P01241 (SEQ ID NO:1)fptiplsrlfdnamlrahrlhqlafdtyqefeeayipkeqkysflqnpqtslcfsesiptpsnreetqqksnlellrisllliqswlepvqflrsvfanslvygasdsnvydllkdleegiqtlmgrledgsprtgqifkqtyskfdtnshnddallknygllycfrkdmdkvetflrivqcrsvegscgf            ↑

This mutated pituitary derived hGH, regardless of the expression systemused to produce this polypeptide, can then be glycosylated orglycoconjugated (see WO 03/31464). Preferably, the mutated pituitaryderived hGH is glycoPEGylated, wherein a polyethylene glycol (PEG)moiety is conjugated to the mutated pituitary derived hGH polypeptidevia a glycosyl linkage (see WO 03/31464, incorporated herein byreference). FIG. 3 describes the GlycoPEGylation of an hGH N-linkedglycan mutant produced in either Sf9 insect cells or mammalian cells.GlycoPEGylation of the mutated pituitary derived hGH is expected toresult in improved biophysical properties that may include but are notlimited to improved half-life, improved area under the curve (AUC)values, reduced clearance, and reduced immunogenicity.

Example 2

An alternative approach is to create an O-linked glycosylation site intothe pituitary derived hGH polypeptide. This O-linked glycosylation sitemay then be used as a site on which the mutated hGH polypeptide can beglycoPEGylated using a GalNAcT₂ enzyme or the like. One or moreadditional transferases may then be used to add glycans orglycoconjugates to that site. Preferably, the mutated pituitary derivedhGH polypeptide is glycoPEGylated. FIG. 4 describes the glycoPEGylationof an hGH O-linked glycan mutant produced in Escherichia coli.

Example 3

As identified by the crystal structure of hGH and its receptor, theprotein loop regions on pituitary derived hGH are best suited formutation to introduce a glycosylation site (FIG. 5). Specifically, thenucleotide sequence that encodes amino acids 1-6 (FPTIPL; SEQ ID NO:10),amino acids 48-52 (PQTSL; SEQ ID NO:11), amino acids 59-64 (PTPSNR; SEQID NO:12), amino acids 133-139 (PRTGQIF; SEQ ID NO:13), amino acids133-145 (PRTGQIFKQTYSK; SEQ ID NO:14), or amino acids 139-142 (FKQT; SEQID NO:15) of the wild-type pituitary derived hGH amino acid sequence(see Table 5 and FIG. 1) can be mutated so that either an N-linked or anO-linked glycosylation site is introduced into the resulting mutatedpituitary-derived hGH polypeptide.

FIG. 6 illustrates six (6) of these introduced O-linked glycosylationsites. The arrows in FIG. 6 each represent the threonine residue onwhich O-linked glycosylation will occur in the GH-N O-linked glycan hGHmutant.

FIG. 7 and FIG. 8 each illustrates two additional GH-N O-linked glycanhGH mutants.

Example 4

This example describes amino acid sequence mutations introducingO-linked glycosylation sites, i.e., serine or threonine residues, into apreferably proline-containing site of a wild-type human growth hormonesequence or any modified version thereof.

N-terminal Mutations

In the N-terminal mutants, the N-terminus of a wild-type hGH, FP²TIP⁵LS;SEQ ID NO:16, is replaced with either MXnTP²TIP⁵LS or MAPTSSXnP²TIP⁵LS.Preferred examples include: MVTPTIPLS; SEQ ID NO:17 MQTPTIPLS; SEQ IDNO:18 MAPTSSPTIPLS; SEQ ID NO:19 MAPTSSSPTIPLS SEQ ID NO:20 (IL-2N-terminus); MPTTFPTIPLS; SEQ ID NO:21 MPTSSPTIPLS; SEQ ID NO:22MPTSSSPTIPLS; SEQ ID NO:23Internal Mutation Site 1

In this type of mutants, the N-terminus of a wild-type hGH, FP² TIP⁵LS;SEQ ID NO:24, is replaced with ZmP²T XnBoP⁵LS. Preferred mutationsinclude: MFPTQIPLS; SEQ ID NO:25 MFPTSIPLS; SEQ ID NO:26 MFPTSSPLS; SEQID NO:27 MTPTQIPLS; SEQ ID NO:28 MFPTTTPLS; SEQ ID NO:29Internal Mutation Site 2

In this type of mutants, the amino acid sequence surrounding p³⁷,AYIP³⁷KEQKY; SEQ ID NO:30, is replace with AZmJqP³⁷OrXnBoΔpY, where atleast one of Z, J, O, X, and B is independently selected from either Thror Ser; Δ may include Lys (K) and X may be Asp (D). Preferred examplesinclude: AYIP³⁷TQGAY; SEQ ID NO:31 AYIP³⁷TSSSY; SEQ ID NO:32AQITP³⁷TEQKY; SEQ ID NO:33 AYIP³⁷TEQSY; SEQ ID NO:34Internal Mutation Site 3

In this type of mutants, the amino acid sequence surrounding p⁴⁸,LQNP⁴⁸QTSLC; SEQ ID NO:35, is replaced with LZmJqP⁴⁸OrXnBoLC, where atleast one of Z, J, O, and X are independently selected from either Thror Ser. Preferred examples include: LQTP⁴⁸QTSLC; SEQ ID NO:36LQNP⁴⁸TTSLC; SEQ ID NO:37Internal Mutation Site 4

In this type of mutants, the amino acid sequence surrounding p⁵⁹,SESIP⁵⁹TPNREET; SEQ ID NO:38, is replaced with SZmUsJqP⁵⁹TPOrXnBoΔrT,where at least one of Z, J, O, B, Δ, U, and X is independently selectedfrom either Thr or Ser; B, Δ, and Z may include charged amino acids.Preferred examples include: SESTP⁵⁹TPNREET; SEQ ID NO:39 SSSTP⁵⁹TPNREET;SEQ ID NO:40 SESIP⁵⁹TPNTEET; SEQ ID NO:41 SESIP⁵⁹TPNTQET; SEQ ID NO:42SESIP⁵⁹TPTQGAT; SEQ ID NO:43 SESIP⁵⁹TPTESST; SEQ ID NO:44SQSTP⁵⁹TPNREET; SEQ ID NO:45 SQSTP⁵⁹TPNQEET; SEQ ID NO:46SESTP⁵⁹TPTSSST; SEQ ID NO:47Internal Mutation Site 5

In this type of mutants, the amino acid sequence surrounding p⁸⁹,SWLEP⁸⁹VQFLRS; SEQ ID NO:48, is replaced with SZmUsJqP⁸⁹OrXnBoΔrλtS,where at least one of Z, U, J, O, B, and X is independently selectedfrom either Thr or Ser; J and λ may include charged amino acids.Preferred examples include: SWLEP⁸⁹TQGLRS; SEQ ID NO:49 SWLEP⁸⁹TQGATS;SEQ ID NO:50 SSQTP⁸⁹VQFLRS; SEQ ID NO:51 SWLEP⁸⁹TSSLSS; SEQ ID NO:52SMVTP⁸⁹VQFLRS; SEQ ID NO:53Internal Mutation Site 6

In this type of mutants, the amino acid sequence surrounding P¹³³,EDGSP¹³³RTGQIF; SEQ ID NO:54, has been replace withEZmUsJqP¹³³OrXnBoΔrλtF, where at least one of Z, U, J, O, B, and X isindependently selected from either Thr or Ser. Preferred examplesinclude: EDGSP¹³³TTGQIF; SEQ ID NO:55 EDGSP¹³³NTGQIF; SEQ ID NO:56EDGSP¹³³TQGQIF; SEQ ID NO:57 EDGSP¹³³TVGQIF; SEQ ID NO:58EDGSP¹³³TTTQIF; SEQ ID NO:59 EDGSP¹³³TSSQIF; SEQ ID NO:60EDGSP¹³³TTQGIF; SEQ ID NO:61 EDGSP¹³³QTGQIF; SEQ ID NO:62EDGTP¹³³NTGQIF; SEQ ID NO:63 EDQTP¹³³NTGQIF; SEQ ID NO:64Internal Mutation Site 7

In this type of mutants, the amino acid sequence surrounding p¹⁴⁰,GQIFK¹⁴⁰QTYS; SEQ ID NO:65, is replace with GZmUsJqΔr¹⁴⁰OrXnBoS, whereat least one of Z, U, J, O, B, and X is independently selected fromeither Thr or Ser. Preferred examples include: GQIFN¹⁴⁰QTYS; SEQ IDNO:66 GQIFN¹⁴⁰ITYS; SEQ ID NO:67 GQIFP¹⁴⁰QTSS; SEQ ID NO:68GQIFP¹⁴⁰TTTS; SEQ ID NO:69 GQITP¹⁴⁰QTYS; SEQ ID NO:70 GQIFT¹⁴⁰QTYS; SEQID NO:71 GQIST¹⁴⁰QTYS; SEQ ID NO:72 GQIPT¹⁴⁰TTYS; SEQ ID NO:73C-terminal Mutations

In this type of mutants, the amino acid sequence at the C-terminus of awild-type hGH, VEGSCG¹⁹⁰F; SEQ ID NO:74, is replaced withVEGSCG¹⁹⁰PXnBoZmUsP, where at least one of Z, U, B, and X isindependently selected from either Thr or Ser. Preferred examplesinclude: VEGSCGPTTTP; SEQ ID NO:75 VEGSCGPTSSP; SEQ ID NO:76VEGSCGPTQGAMP; SEQ ID NO:77 VEGSCGPTTIP; SEQ ID NO:78 VEGSCGPMVTP; SEQID NO:79

In all above cases, X, Z, B, A, J, U, O, and A are independentlyselected from E (glutamate), any uncharged amino acid or dipeptidecombination including M, F, MF, and the like; m, n, o, p, q, r, s, and tare independently selected from integers from 0 to 3. In all cases, theN-terminal Met may be present or absent on any hGH mutant. The numberingof the amino acid residues is based on the initial unmodified sequencein which the left most residue is numbered 1. The numbering ofunmodified amino acids remains unchanged following the modification.More than one of the above described sequence modifications may bepresent in a hGH mutant of the present invention.

Example 5

This example illustrates one embodiment of the present invention,wherein a mutant hGH with the P¹³⁴TINT glycosylation site motif firstundergoes glycoPEGylation with the use of GalNAcT₂ and UDP-GalNAc incombination with ST6GalNAc-I and CMP-SA-PEG (30 kDa), then chemicalPEGylation, resulting in a hGH with protease resistance.

Example 6

This example illustrates one embodiment of the present invention,wherein a mutant hGH with the P¹³⁴TQGAM glycosylation site motif firstundergoes chemical PEGylation, then glycoPEGylation with the use ofGalNAcT₂, core-1-GalT₁, ST3Gal₁ in combination with CMP-SA-PEG-Cys (40kDa), thereby resulting in a hGH with protease resistance.

Example 6

This example illustrates one embodiment of the present invention,wherein a mutant hGH with the P¹³⁴TINT glycosylation site motif firstundergoes glycoPEGylation with the use of GalNAcT₂ and UDP-GalNAc incombination with core-1-GalT₁/UDP-Gal and ST3Gal₁/CMP-SA-PEG (40 kDa),then chemical PEGylation, resulting in a hGH with protease resistance.It should be noted that only one size for m should be used for anysingle reaction and product.

Example 7

This example illustrates one embodiment of the present invention,wherein hGH mutants are expressed in E. coli JM 109 and W3110. Miniprepplasmid DNA from hGH mutants were used to transform E. coli JM109 andW3110, respectively. The resulting transformants were inoculated into 2ml Martone LB containing 10 μg/ml kanamycin and grown overnight at 37°C. as starter cultures. One half milliliter of the starter culture weretransferred to 50 ml Martone LB containing 10 μg/ml kanamycin andallowed to grow at 37° C./250 rpm until OD₆₀₀ reached 0.8˜1.0. IPTG wasthen added to a final concentration of 1 mM and the induction wasperformed for 5 hrs at 37° C. with a reduced agitation rate of 150 rpm.One hundred microliters of each culture before and after induction weretaken and the bacteria were harvested by centrifugation in a bench topcentrifuge. Each pellet was resuspended in 20 μl H₂O plus 20 μl2×SDS-PAGE sample buffer and 4 μl DTT stock solution (1 M). The cellswere lysed and the proteins denatured by heating at 95° C. for 5 min.Ten microliters from each sample were loaded onto a 14% acrylamideTris-Glycine precast gels. The electrophoresis was performed at constantvoltage (125 V) until the dye moved to the bottom of the gel. The gelwas stained with Simple blue Safestain (see FIG. 36).

Induction of hGH Mutant Expression in Shake Flask

Small Scale Induction for glycoPEGylation Screening

Plasmid DNA for each hGH mutant were transformed into E. coli W3110.Several colonies from each transformation plate were inoculated into 20ml Martone LB containing 10 μg/ml kanamycin and grown at 37° C./250 rpmfor ˜6 hrs as starter culture. Ten milliliters of the starter culturewere then transferred to 500 ml Martone LB containing 10 μg/ml kanamycinand grown at 37° C./250 rpm until OD₆₀₀ reached 0.8-1.0. IPTG was addedto a final concentration of 1 mM and the induction was performedovernight at 37° C. with 150 rpm. For some of the mutants, the hGHmutant expression was analyzed by SDS-PAGE as described above.

Induction at 1 L Scale

The starter culture was inoculated using a scraping from a glycerolstock of each individual mutant. The bacteria were incubated at 37° C.,135 rpm overnight. Ten milliliters of starter culture were transferredto 1 L Martone LB containing Kanamycin (10 μg/ml) in 2 L-shaking flask(baffled or non-baffled) and grown at 37° C., 250 rpm until OD₆₀₀ of0.8˜1.0 (around 3 hrs). IPTG was added to a final concentration of 1 mMand the induction was performed at 37° C., 135 rpm for 15 hrs. TheMartone LB media contained 1% Martone B-1, 0.5% Marcor Yeast Extract, 1%NaCl. The pH was adjusted to 7.0 by adding 3.25 ml 1N NaOH to 1 Lmedium.

Induction of hGH Mutant Expression in 10-L Fermentor

8 L of media were prepared containing 1% Martone B-1, 0.5% Marcor YeastExtract, 1% NaCl. The pH was brought to 7.0 after autoclaving and thenthe acid/base valves were turned off. Kanamycin at a final concentrationof fifteen μg/ml was added. The growth was performed under full air flowat 37° C., 250 rpm until OD₆₀₀ reached 0.5-1.0 using 180 ml overnightculture as the inoculum. The protein expression was induced by theaddition of 1 mM IPTG (final concentration) under ½ air flow rate at 125rpm for 24-30 hrs. Cells were harvested by centrifugation.

Induction of hGH Mutant Expression in 150-L Fermentor

The parameters for the 150-L fermentor were designed to mimic those ofthe above mentioned 10-L fermentor. Kanamycin was used through allstages of culture at a final concentration of 10 μg/ml. A 200 ml shakeflask seed culture was inoculated with 100 μl from a thawed 1 mlglycerol stock and grown at 37° C./135 rpm for 16-18 hrs. The shakeflask seed culture was then transferred to 15-L seed fermentorcontaining 10 L media and grown overnight. Two to ten liters of the seedculture, preferably 2 L, were used to inoculate up to 98 L productionculture (with combined volume of 100 L in a 150-L fermentor). Thecultures were grown to an OD₆₀₀ of 0.6 to 3.0, preferably 0.6, at 37° C.and 25% pO₂ under cascade mode. The expression of each hGH mutant wasinduced by the addition of IPTG at a final concentration of 1 mM. Thefermentation was continued at an agitation of 75 rpm and 10% pO₂(cascade mode) at 37° C. for 24 hrs. Cells were harvested using a discstack centrifuge.

Preparation of Inclusion Bodies (IBs) for hGH Mutants

Small Scale IB Preparation for glycoPEGylation Screening

Induced cultures were harvested by centrifugation at 4° C./5000 rpm for15 min. The pellets obtained were resuspended in a buffer containing 20mM Tris-HCl, pH 8.5, 5 mM EDTA. The resuspended bacterial cells weredisrupted by passing through a microfluidizer twice at ˜16,000 Psi. TheIBs were collected by centrifugation at 4° C., 5000 rpm for 10 min andwashed twice, first with 35 ml and second with 10 ml washing buffercontaining 20 mM Tris-HCl, pH 8.5, 5 mM EDTA, 100 mM NaCl, 1% TritonX-100 and 1% sodium deoxycholate. The IBs were finally resuspended in 30ml H₂O and aliquoted equally into 3 50-ml conical tubes. The IBs werecollected and stored at −80° C. Five to ten microliter aliquots wereanalyzed on 14% acrylamide Tris-Glycine precast gels as described above(see FIG. 37).

Large Scale IB Preparation for glycoPEGylation

The induced cultures were harvested by centrifugation at 4° C. with 5000rpm for 10 min. The pellets obtained were resuspended (˜4 g/100 ml) in abuffer containing 20 mM Tris-HCl, pH 8.5, 5 mM EDTA. The resuspendedbacterial cells were disrupted by passing through a microfluidizertwice. The IBs were collected by centrifugation at 4° C., 4150 rpm for10 min and washed repeatedly as shown in Table 6 below. TABLE 6Procedures of scaled-up IB washing Collection (g) × Step Wash time (min)Note 1 150 ml wash 5000 g × 10 IBs from 9 L culture buffer min 2 150 mlwash 5000 g × 10 Buffer min 3 150 ml H₂O 5000 g × 10 Top loose layerremoved min 4 150 ml Wash 5000 g × 10 buffer min 5 150 ml Wash 5000 g ×10 buffer min 6 150 ml H₂O 5000 g × 10 Top loose layer removed min 7 150ml Wash 5000 g × 10 buffer min 8 150 ml H₂O 5000 g × 10 Top loose layerremoved minRefolding of Inclusion Bodies for hGH Mutants:hGH Mutants Refolded in a Buffer Containing Redox Couple

150 mg washed IBs were first solubilized in 7.5 ml buffer containing 6 Mguanidine HCl, 5 mM EDTA, 100 mM NaCl, and 50 mM Tris-HCl (pH 8.5) byconstant stirring at room temperature. The solubilized IBs were thendiluted slowly into a refolding buffer with combined volume of 150 mlcontaining, 0.5 M L-Arg+, 250 mM NaCl, 10 mM KCl, 0.05% PEG 3350, 50 mMMES, 1 mM GSH, 0.1 mM GSSG, 0.3 mM lauryl maltoside, pH approximately10.5. The refolding was performed at 4° C. for 2.5 hrs with slow,constant stirring then dialyzed overnight against 2 L buffer containing50 mM NaOAc, pH 4.0, 50 mM NaCl and 10% glycerol. The pH of the dialysisbuffer can be varied from 4.0 to 7.4, but preferably 7.4.

hGH Mutants Refolded by Alkaline Solubilization

The refolding of IBs was carried out in ˜200 mg aliquots using thefollowing procedure (see FIG. 38):

-   -   1. Solubilization of each IB aliquot at a ratio of 5 mg/ml in        ice-cold buffer containing 100 mM Tris, pH 12.5, 2 M urea by        constant stirring until no significant pellets were visible;    -   2. Dilution by adding 4 volumes of cold water batchwise without        stirring;    -   3. Adjusting pH to 8.5 using 1 N HCl with constant, mild        stirring;

The refolded aliquots were pooled and filtered through a 0.45 μmmembrane prior to loading onto DEAE ion exchange chromatography.

Example 8

Acute Toxicity Study of Glycopegylated hGH Compounds Administered byEither the Intravenous or Subcutaneous Route to Rats

The formulation was used as received for dose administration. Allanimals were dosed once, intravenous or subcutaneous or subcutaneous, onStudy Day 1. Dose volume was dependent upon final Dosage Concentrationto yield a final dose per animal of 90 μg/rat (see Table 7 below). TABLE7 Dosage Group Test Route of Level Number of Number ArticleAdministration (μg/rat) Males 1 AA subcutaneous 90 12 2 AV subcutaneous90 12 3 AW subcutaneous 90 12 4 AS subcutaneous 90 12 5 AQ subcutaneous90 12 6 AP subcutaneous 90 12 7 AU subcutaneous 90 12 8 AO subcutaneous90 12 9 AR subcutaneous 90 12 10 AA intravenous 90 12 11 AV intravenous90 12 12 AW intravenous 90 12 13 AS intravenous 90 12 14 AQ intravenous90 12 15 AP intravenous 90 12 16 AU intravenous 90 12 17 AO intravenous90 12 18 AR intravenous 90 12

Blood for evaluation of pharmacokinetics was collected from 4rats/subgroup according to Table 8 below. TABLE 8 Number ofsamples/group collected Intravenous at each time-point (postdose)Subgroup/ 5 min, 1 hour, 8 15 min, 2 hour, 12 30 min, 4 hour, testarticle hour, 48 hour hour, 72 hour 24 hour, 96 hour A 4 B 4 C 4 Sub-Number of samples/group collected cutaneous at each time-point(postdose) Subgroup/ 1 hour, 12 hour, 4 hour, 24 hour, 8 hour, 36 hour,test article 48 hour, 96 hour 60 hour 72 hour A 4 B 3 C 3

Blood was processed to serum, frozen (−70° C.±5° C.) and shippedovernight to Neose Technologies, Inc. for concentration analysis. AnELISA was used to measure the concentration of hGH. Pharmacokineticanalysis was then performed.

Example 9

10-Day Study Comparing the Efficacy of Glycopegylated andNon-Glycopegylated Human Growth Hormone Following SubcutaneousAdministration in Hypophysectomized Female Rats

A total of 65 hypophysectomized female Sprague-Dawley rats were assignedto 13 groups (5/group). Animals were administered either 200 μL ofVehicle (a solution of phosphate buffered saline at pH 7.4, 0.3%Pluronic F-68, and 2% Mannitol), 180 μg or 30 μg/dose of the referencecontrol article (P148), or 180 μg of one of the test articles (P241,P242, P249-64, P249-72, P250, P256, P257, P258, P259, or P240) viasubcutaneous injection. All animals were administered a single dose onStudy Day 1, with the exception of animals dosed with 30 μg/dosereference control (P148) which were administered 10 consecutive dailydoses beginning on SD 1. (See Table 9 below) TABLE 9 ProteinConcentration Dose Level Protein Dosage Number of Animal Group Treatment(μg/mL) (μL/dose) (μg/dose) Animals Numbers 1 Vehicle-62 0 200 0 520565-20569 2 AA 1310 137 180 5 20570-20574 3 AA 1310 23 30 520575-20579 4 AV 1130 159 180 5 20580-20584 5 AW 1140 158 180 520585-20589 6 AS-64 970 186 180 5 20590-20594 7 AS-72 1000 180 180 520595-20599 8 AQ 897 200 180 5 20600-20604 9 AP 1030 175 180 520605-20609 10 AU 1620 111 180 5 20610-20614 11 AO 1290 139 180 520615-20619 12 AR 980 184 180 5 20620-20624 13 AX 1190 151 180 520625-20629Different reference control groups (Groups 2 and 3) were established toevaluate the efficacy of the test articles compared to differentinjection frequencies of an established hGH formulation.

Rats were administered a subcutaneous injection once on Study Day 1 forGroups 1, 2, and 4-13 and once daily (approximately the same time eachday, ±1 hour) on Study Day 1-10 for Group 3. Dosing materials weremaintained on wet ice prior to and during dosing. All animals werechecked for general health/mortality twice daily, clinical observationspredose and termination and body weights recorded predose, Study Day 1and daily thereafter.

Blood was collected for IGF-1 evaluation on Study Day-3 (prestudy), 1,2, 4, 7, and 10, stored on wet ice, and centrifuged at ˜3000 rpm for 10min at 4° C. Plasma was transferred to microcentrifuge tubes and storedat −75±10° C. Plasma was shipped to Ani Lytics on dry ice forinsulin-like growth factor 1 (IGF-1) plasma concentration (IGF-1analysis).

A gross necropsy, which included examination of the external surface ofthe body, injection site, all orifices, the cranial, thoracic, andabdominal cavities, and their contents, was performed on survivinganimals on Study Day 11. Organs were weighed as soon as possible afterdissection at scheduled necropsies. Paired organs were weighed together.Moribund animals were subjected to a gross necropsy and protocolspecified-tissues were collected; however no organ weights werecollected. At scheduled termination, the absence of the pituitary glandwas documented. The eyes were fixed in modified Davidson's fixative forapproximately 24 hours and then transferred to 70% alcohol. All grosslesions and protocol-required tissues were preserved in 10% neutralbuffered formalin (NBF).

Body weights, body weight changes, absolute and relative organ weights,and IGF-1 concentration were statistically analyzed. Analysis wasperformed for all test articles against each reference control group(Groups 2 and 3).

Example 10

DEAE Purification

The refolded hGH (2 L) was passed through a 0.2 micron in-line filterand was loaded on a DEAE Sepharose column (200 mL) pre-equilibrated with20 mM tris buffer pH 8.5, 5 mM EDTA, 0.4 M urea and connected to an HPLCsystem that monitored the absorbance at 280 nm. The hGH was eluted witha step gradient from 20 mM tris buffer pH 8.5, 5 mM EDTA to 20 mM trisbuffer pH 8.5, 5 mM EDTA, 50 mM NaCl followed by a linear gradient to 20mM tris buffer pH 8.5, 5 mM EDTA, 2 M NaCl. Fractions were collected ina chilled fraction collector and concentrated to approximately 1 mg/mLusing a centrifugal filter (5 kDa MWCO). Samples were stored at 4° C.

Superdex 200 Purification

The concentrated DEAE-sepharose purified hGH samples (approximately 9mL) were loaded on a Superdex 75 column connected to an HPLC systemmonitoring absorbance at 280 nm. The hGH product was eluted with 50 mMTris, pH 7.4, 20 mM NaCl at a flow rate of 4 mL/min. Fractionscontaining monomeric product were collected and stored at 4° C.

Example 11

TABLE 10 Table of Mutants: In vitro activity (Nb2-11 cell AA sequence ofhGH mutants proliferation) P134TTGQIF Active P134TTAQIF ActiveP134TATQIF Active P134TQGAMF Active P134TQGAIF Active P134TQGQIF ActiveP134TTLYVF Active P134TINTIF Active P134TTVSIF Active I139PTQTYS ActiveI139PTQAYS Active P134TTLQIF Active P134TTVQIF Active P134TTNQIF ActiveP134TTQQIF Active P134TIGQIF Active P134TILQIF Active P134TIVQIF ActiveP134TINQIF Active P134TIQQIF Active P134TIAQIF Active L129ETETPRT ActiveL129VTETPRT Active L129ETQSPRT Active L129VTQSPRT Active L129VTETPATActive L129ETETPAT Active L129ATGSPRT Active L129ETQSPST ActiveL129ETQSPAT Active L129ETQSPLT Active Y43 change to A; ActiveP134TINTIFKQTYS Y43 change to A; Active P134TINTIFKQTA, Y43 change to A;Active P134TINTIANQTA Y43 change to A; Active I139PTQAYS V1TPT ActiveP134TQGAMP Active P134TTTQIF Active P134NTGQIF Active M1FPTEIP ActiveM1FPTVLP Active

Example 12

Preparation of hGH-GalNAc-SA-PEG-30 kDa (Two Sugar Preparation)

The hGH mutant (14.0 mg, 0.63 micromole) was adjusted to 0.001%Polysorbate 80 and concentrated to a volume of 2-3 mL using acentrifugal filter (5 kDa MWCO). The UDP-GalNAc (6.3 micromoles) and 1MMnCl₂ (to a final concentration of 10 mM MnCl₂) were then added to theconcentrated hGH solution. The GalNAcT2 (50 microliters, 105 mU) wasadded, mixed very gently and incubated at room temperature for 18.5 hrs.The addition of GalNAc was determined to be complete by MALDI analysisof the reaction mixture. The CMP-SA-PEG-30 kDa (1.9 micromoles) wasadded to the hGH-GalNAc reaction mixture as a concentrated solution inreaction buffer and ST6GalNAc1 (0.72 mL, 0.7 U) was added with gentlemixing. The reaction mixture was incubated at room temperature for 20hrs and was monitored for extent of PEGylation by SDS PAGE and RP-HPLC.The product, hGH-GalNAc-SA-PEG-30 kDa, was purified using SP Sepharoseand SEC (Superdex 200) chromatography. The purified hGH-GalNAc-SA-PEG-30kDa was concentrated and then formulated. The product was analyzed by aBCA protein assay, SDS-PAGE gels (silver stain), MALDI, SEC foraggregation, and for cell proliferation. (See Table 11 below) TABLE 11SA-PEG-3OkDa Addition GalNAc Addition - (2 sugar product) GalNAcT2Conversion Conversion Yield by hGH mutant Yield (MALDI) RP-HPLCP134TINTIF 100% 83-89% P134TTVSIF 100% 74-85% I139PTQAYS 100% 66-86%P134TQGAMF 100% 86% M1FPTEIP 100% 27% M1FPTVLP 100% 11-18%

Preparation of hGH-GalNAc-Gal-SA-Cys-PEG-40 kDa (Three SugarPreparation)

The hGH mutant (42 mg, 1.9 micromoles) was adjusted to 0.001%Polysorbate 80 and concentrated to a volume of 6-7 mL using acentrifugal filter (5 kDa MWCO). The UDP-GalNAc (19.1 micromoles) wasadded as a concentrated solution in Reaction buffer: 50 mM Tris, 20 mMNaCl, 0.001% Polysorbate 80, 0.02% NaN₃, pH 7.4 (0.2 mL) and theresulting solution was adjusted to 10 mM MnCl₂ (1 M MnCl₂, 0.08 mL). TheGalNAcT2 (200-400 mU) was added, mixed very gently and incubated at roomtemperature for 18.5 hours. The addition of GalNAc was determined to becomplete by MALDI analysis of the reaction mixture. A concentratedsolution of UDP-Gal in Reaction buffer (19 micromoles, 0.1 mL) andCore-1-GalT1 enzyme (0.42 U) were added to the hGH-GalNAc reactionmixture with gentle mixing and the resulting solution was incubated for16.5 hrs at room temperature. The addition of Gal was determined to becomplete by MALDI analysis of the reaction mixture. TheCMP-SA-cys-PEG-40 kDa (3.8 micromoles) was added to the hGH-GalNAc-Galreaction mixture as a concentrated solution in reaction buffer (0.8 mL)and ST3 Gal₁ enzyme (1.68 U) was added with gentle mixing. The reactionmixture was incubated at room temperature for 20-65 hrs and wasmonitored for extent of PEGylation by SDS PAGE and RP-HPLC. The product,hGH-GalNAc-Gal-SA-cys-PEG-40 kDa, was purified using SP Sepharose andSEC (Superdex 200) chromatography. The purifiedhGH-GalNAc-Gal-SA-cys-PEG-40 kDa was concentrated and then formulated.The product was analyzed by a BCA protein assay, SDS-PAGE gels (silverstain), MALDI, SEC for aggregation, and for cell proliferation. (SeeTable 12 below) TABLE 12 SA-PEG-cys- 4O kDa GalNAc Addition Addition-Gal Addition (3 sugar GalNAcT2 Core-1GalT1 product) ConversionConversion Conversion Yield Yield Yield hGH mutant (MALDI) (MALDI)RP-HPLC P134TINTIF 100% 100% 25-45% P134TTVSIF 100% 100% (40% SDS- PAGEI139PTQAYS 100% 100% 23-24% P134TQGAMF 100% 100%   32% M1FPTEIP 100%100% (20% SDS- PAGE MIFPTVLP 100% 100% 20-40% Y43 change to A; 100% 100%28-32% P134TINTIFKQTYS Y43 change to A; 100% 100% 22-24% P134TINTIFKQTA,Y43 change to A; 100% 100%   50% P134TINTIANQTA Y43 change to A; 100%100% 28-38% I139PTQAYS

SP Sepharose Chromatography: The hGH-GalNAc-SA-PEG-30 kDa andhGH-GalNAc-Gal-SA-Cys-PEG-40 kDa were purified using an SP Sepharosecolumn (20 mL) connected to an HPLC system that monitored the absorbanceat 280 nm. The hGH reaction mixtures were diluted into 5 volumes of cold25 mM sodium acetate (pH 4.2) and were injected onto the column. Theproduct was eluted at a flow rate of 5.0 mL/min. using a NaCl gradient.Product containing fractions were collected, neutralized to pH>7 with 1M Tris, pH 8 and concentrated to approximately 1.0 mL using acentrifugal filter, 5 kDa MWCO. Samples were stored at 4° C.

Size Exclusion Chromatography: The hGH-GalNAc-SA-PEG-30 kDa andhGH-GalNAc-Gal-SA-cys-PEG-40 kDa were purified on a Superdex 200 columnusing an HPLC that monitored absorbance at 280 nm. The concentratedSP-sepharose purified samples (approximately 1 mL) were loaded and theproducts were eluted with PBS (pH 7.4) at a flow rate of 1-2 mL/min.Product containing fractions were collected and stored at 4° C.

SDS PAGE Analysis: The 4-20% polyacrylamide gradient slab gels wereused. Samples (approximately 10 mcg of protein) were mixed with 10microliters SDS Sample Buffer containing 0.1 M DTT, and heated at 85° C.for 6 min, unless otherwise noted. Gels were run at a constant voltageof 125 V for 1 hr 50 min. After electrophoresis, the proteins werestained with a colloidal stain solution for 2-24 hours at roomtemperature as necessary to visualize the protein or with a silver stainkit.

Example 13

Cell Proliferation Assay

An Nb2-11 cell proliferation assay was used to determine the in vitroactivity of the hGH mutants and glycoPEGylated hGH mutants. The assaywas based that described in Patra, A. K., Mukhopadhyay, R., Mukhiga, R.,Krishnan, A., Garg, L. C. and Panda, A. K. Optimization of inclusionbody solubilization and renaturation of recombinant human growth hormonefrom Escherichia coli. Protein Expr. Purif. 18, 182-192 (2000). Nb2-11cells were grown in Fischer's media with lactogen-free 10% horse serumand 10% fetal bovine serum, 2 mM glutamine, 0.05 mM 2-mercaptoethanol ina humidified chamber at 37° C., 5% CO₂. Nb2-11 cells were washed twicewith PBS to remove the growth media and then resuspended and incubatedin Arrest Media containing Fischer's media with lactogen-free 10% horseserum and 1% fetal bovine serum, 2 mM glutamine, 0.05 mM2-mercaptoethanol at 37 C, 5% CO₂ for 24 h. After incubating the cellsunder starving conditions, Nb2-11 cells were dispensed into a 96-wellplate at a density of 5×10⁴ cells/mL. hGH samples were sterile filteredand diluted at different concentrations in dilution medium (Fischer'smedia with lactogen-free 10% horse serum, 2 mM glutamine, 0.05 mM2-mercaptoethanol and 2 mM HEPES). The diluted hGH samples were added tothe plated cells in 6 replicates. The cells were then incubated for 72hours in a humidified chamber at 37° C. with 5% CO₂. After incubatingfor 72 hours, MTS reagent was added to each plate as a colorimetricindicator of cell density. The reduced by-product of MTS was measuredcalorimetrically at an absorbance of 490 nm using an ELISA Plate. Priorto addition of the MTS, Nb2-11 cells, maintained separately under normalgrowth conditions, were dispensed at known cell densities into reservedempty wells on each plate were used to generate a standard curve. Usinga linear regression analysis of the standard curve, the cell density ateach sample concentration was interpolated from the average of the 6replicates.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1. An isolated nucleic acid comprising a polynucleotide sequence encoding a mutant human growth hormone, wherein said mutant human growth hormone comprises a member selected from a protease recognition site comprising a proteolysis resistant mutation not present in wild type human growth hormone, an N-linked glycosylation site mutation not present in wild type human growth hormone, an O-linked glycosylation site mutation not present in wild-type human growth hormone, and combinations thereof.
 2. The nucleic acid of claim 1, wherein said wild-type human growth hormone has the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2.
 3. The nucleic acid of claim 1, wherein said O-linked glycosylation site mutation is proximate a proline residue.
 4. The nucleic acid of claim 3, wherein said proline residue is located at a position which is a member selected from position 2, 5, 37, 48, 59, 89, 113, 140, 190, and combinations thereof, of a member selected from SEQ ID NO:1 and SEQ ID NO:2.
 5. The nucleic acid of claim 1, wherein the mutant human growth hormone comprises an amino acid sequence which is a member selected from SEQ ID NO:3, 4, 5, 6, 7, 8, 9, 80, 81, 82, 83, 84, 85, 86, 87, 88 and
 89. 6. The nucleic acid of claim 1, wherein the mutant human growth hormone comprises more than one glycosylation site mutation.
 7. An expression cassette comprising the nucleic acid of claim
 1. 8. A cell comprising the nucleic acid of claim
 1. 9. A mutant human growth hormone, comprising a mutation which is a member selected from a protease recognition site comprising a proteolysis resistant mutation not present in wild type human growth hormone, an N-linked glycosylation site mutation not present in wild type human growth hormone, an O-linked glycosylation site mutation not present in wild-type human growth hormone, and combinations thereof.
 10. The mutant human growth hormone of claim 9, wherein said wild-type human growth hormone has an amino acid sequence which is a member selected from SEQ ID NO:1 and SEQ ID NO:2.
 11. The mutant human growth hormone of claim 9, wherein said glycosylation site mutation is proximate a proline residue.
 12. The mutant human growth hormone of claim 11, wherein said proline residue is located at a position which is a member selected from position 2, 5, 37, 48, 59, 89, 113, 140, 190, and combinations thereof, of a member selected from SEQ ID NO:1 and SEQ ID NO:2.
 13. The mutant human growth hormone of claim 9, comprising an amino acid sequence which is a member selected from SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 80, 81, 82, 83, 84, 85, 86, 87, 88, and
 89. 14. The mutant human growth hormone of claim 9, wherein the mutant human growth hormone comprises more than one glycosylation site mutation.
 15. The mutant human growth hormone of claim 9, comprising a water-soluble polymer attached to said glycosylation site mutation through a glycosyl linker.
 16. The mutant human growth hormone of claim 15, wherein said glycosyl linker is an intact glycosyl linker.
 17. The mutant human growth hormone of claim 16, wherein said intact glycosyl linker is a member selected from a galactosyl, an N-acetylgalactosyl and a sialic acid residue.
 18. The mutant human growth hormone of claim 15, wherein said water-soluble polymer is poly(ethylene glycol).
 19. The mutant human growth hormone of claim 15, wherein said O-glycosylation site mutation comprises an amino acid which is member selected from threonine and serine and said glycosyl linker is covalently attached to a member selected from said threonine and said serine.
 20. The mutant human growth hormone of claim 9, further comprising a water-soluble polymer covalently bound to a member selected from an amino acid which is said protease resistant mutation, an amino acid residue proximate said amino acid which is said protease resistant mutation and combinations thereof.
 21. The mutant human growth hormone of claim 20, wherein said water-soluble polymer is a poly(ethylene glycol).
 22. A pharmaceutical formulation comprising a mutant human growth hormone according to claim 9 and a pharmaceutically acceptable carrier.
 23. A method of treating a subject in need of supplementation of endogenously produced human growth hormone, said method comprising administering to said subject a therapeutically effective amount of a mutant human growth hormone according to claim
 9. 24. A method for making a mutant human growth hormone comprising a mutation which is a member selected from a protease recognition site comprising a proteolysis resistant mutation not present in wild type human growth hormone, an N-linked glycosylation site mutation not present in wild type human growth hormone, an O-linked glycosylation site mutation not present in wild-type human growth hormone, and combinations thereof, said method comprising: (a) transfecting a cell capable of expressing said mutant human growth hormone with a nucleic acid encoding said mutant human growth hormone; and (b) expressing said mutant human growth hormone.
 25. The method according to claim 24, further comprising: (c) contacting said mutant human growth hormone with a sugar donor and an enzyme for which said sugar donor is a substrate under conditions appropriate to transfer a sugar moiety from said donor to said glycosylation site.
 26. The method according to claim 25, wherein said sugar moiety is a modified sugar moiety.
 27. The method according to claim 26, wherein said sugar moiety is modified by a water-soluble polymer covalently attached thereto.
 28. The method according to claim 27, wherein said water-soluble polymer is a poly(ethylene glycol).
 29. The method according to claim 25, wherein said sugar moiety is a member selected from a galactosyl, N-acetylgalactosyl and a sialic acid moiety.
 30. The method according to claim 24, further comprising: (d) contacting said mutant human growth hormone with an activated water-soluble polymer under conditions appropriate to form a covalent bond between a water-soluble polymer moiety of said activated water-soluble polymer and an amino acid residue of said mutant human growth hormone.
 31. The method according to claim 30, wherein said amino acid residue is a member selected from said protease resistant mutation, an amino acid residue proximate to said mutation and combinations thereof.
 32. The method of claim 24, wherein said wild-type human growth hormone has an amino acid sequence which is a member selected from SEQ ID NO:1 and SEQ ID NO:2.
 33. The method of claim 24, wherein said O-glycosylation site mutation is proximate a proline residue.
 34. The method of claim 33, wherein the proline residue is located at position which is a member selected from 2, 5, 37, 48, 59, 89, 113, 140, 190, and combinations thereof, of a member selected from SEQ ID NO:1 and SEQ ID NO:2.
 35. The method of claim 24, wherein the mutant human growth hormone comprises an amino acid sequence which is a member selected from SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 80, 81, 82, 83, 84, 85, 86, 87, 88, and
 89. 36. The method of claim 24, wherein the mutant human growth hormone comprises more than one glycosylation site mutation.
 37. The method of claim 33 wherein said O-glycosylation mutant is a member selected from threonine and serine.
 38. The method according to claim 25, said method further comprising: (d) prior to step (c), said mutant human growth hormone is isolated. 